Mammalian Apolipoprotein Research Articles

Apolipoprotein B mRNA Editing is Modulated by Thyroid Hormone Analogs but not Growth Hormone Administration in the Rat

Recent work has demonstrated that the unique post-transcriptional editing reaction which modifies mammalian apolipoprotein (apo) B100 mRNA, producing an in-frame stop codon in the modified (apo B48) transcript, is modulated in vivo in the rat liver by thyroid hormone (T3). We now report the results of studies undertaken to examine the effects of two synthetic T3 analogs and GH on apo B gene expression together with their effects on hepatic apo A-I, A-IV, C-III, and malic enzyme (ME)mRNAs. The T3 analogs were previously shown to exhibit similar binding to the hepatic nuclear T3 receptor (50% and 38% of native T3) but differing biopotency (18 and less than 3% of native T3). Apo B100 mRNA editing, determined by differential hybridization of polymerase chain reaction amplified apo B cDNA, demonstrated 50-56% unmodified (apo B100) mRNA in control and hypothyroid animals and this proportion was unaltered by GH (61% B100 mRNA), despite a reduction in apo B100 synthesis. Both T3 analogs altered apo B mRNA editing (12-16% B100 mRNA) and no apo B100 synthesis was detectable in vivo. Additionally, both T3 analogs produced a 4- to 10-fold induction in hepatic apo A-I and A-IV mRNA abundance, similar to the effects of native T3. GH produced no alteration in apo A-I or A-IV mRNA abundance and neither T3 analog, GH, or native T3 produced a change in apo C III mRNA abundance.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonimmunochemical quantitation of mammalian apolipoprotein A-I in whole serum or plasma by nonreducing gel electrophoresis.

A rapid and convenient method for the quantitation of mammalian apoA-I has been developed. The method involves nonreducing sodium dodecyl sulfate gel electrophoresis and Coomassie blue staining, and takes advantage of the relative abundance of apoA-I in whole serum or plasma. ApoA-I was sufficiently resolved to allow quantitation by laser densitometry or spectrophotometry. The assay was linear from 0.25 to 4.0 micrograms of apoA-I. Analytic recovery was 98%. Within-assay variability was 3.1% and between-assay variability was 7.5%. A high degree of positive correlative (r = 0.98) was observed with a human apoA-I radioimmunoassay. For several species investigated, the apoA-I values obtained correlated strongly and positively with high density lipoprotein cholesterol values. When applied to a study of nutritional perturbation in the Mongolian gerbil, the method detected sensible and significant changes in serum apoA-I that paralleled changes in HDL cholesterol.

Primary structure of apolipophorin-III from the migratory locust, Locusta migratoria. Potential amphipathic structures and molecular evolution of an insect apolipoprotein.

The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Locusta migratoria, has been deduced from the sequence of its cloned cDNA. The mature hemolymph protein consists of 161 amino acids. Optimized alignments of this protein with apolipophorin-III from the tobacco hornworm, Manduca sexta, disclosed an overall sequence identity of only 29%, even though the two proteins are functionally equivalent. The L. migratoria sequence is composed of 12 repeating peptides that are variable in length. Six amphipathic helical segments of varying length were identified in each protein using a newly described algorithm for detecting such secondary structures. The degree of sequence identity between the two insect apoproteins is considerably less than that observed among orthologous mammalian apolipoproteins. However, calculation of the rates of synonymous and nonsynonymous nucleotide substitutions indicates that the insect genes may be evolving at rates similar to the mammalian apolipoprotein genes. Further comparative analyses of insect and mammalian apolipoproteins should provide insights about the limits of sequence diversity tolerated by their predicted amphipathic helical domains.

Primary structure and comparative sequence analysis of an insect apolipoprotein. Apolipophorin-III from Manduca sexta.

The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Manduca sexta, was determined by a combination of cDNA and protein sequencing. The mature hemolymph protein consists of 166 amino acids. The cDNA also encodes for an amino-terminal extension of 23 amino acids which is not represented in the mature hemolymph protein. The existence of a precursor protein was confirmed by in vitro translation of fat body mRNA. Computer-assisted comparative sequence analysis revealed the following points: 1) the protein is composed of tandemly repeating tetradecapeptide units with a high potential for forming amphiphilic helical structures. Compared to mammalian apolipoproteins the repeat units in the insect apolipoprotein show considerable length variability; 2) the sequence has a striking resemblance to several human apolipoproteins including apoE, AIV, AI, and CI. However, the homology seems to be entirely functional since, although the insect and mammalian apoproteins contain very similar types of amino acid residues, the actual degree of sequence identity is quite low. Whether the mammalian and insect apoproteins are derived from a common ancestral amphiphilic helix forming, lipid-binding protein, or arose by convergent evolution can not be determined at present. This represents the first complete amino acid sequence for an insect apolipoprotein.

Genetic factors controlling structure and expression of apolipoproteins B and E in mice.

We report the identification and partial characterization of polymorphisms among inbred strains of mice affecting several aspects of the expression of apolipoproteins B and E (apoB and apoE), the major proteins of low density lipoproteins (LDL) and very low density lipoproteins (VLDL). These polymorphisms include differences in the levels of the lipoproteins and apolipoproteins on both chow and high fat diets, differences in their response to a high fat diet challenge, and differences in the relative levels of the two molecular weight species of apoB. Although most strains exhibited a large increase in plasma LDL and VLDL in response to a high fat diet, the levels of apoB and apoE mRNA were either unaffected or, in some cases, decreased slightly. Also, the levels of apoB and apoE mRNA were not correlated among strains with the levels of the apolipoproteins in plasma, suggesting that genetic control occurs primarily at the level of lipoprotein catabolism. Elucidation of the precise mechanisms involved in the differences will require genetic analysis. Toward this end, we have identified DNA polymorphisms for apoB and apoE and have used these in segregation analysis to determine the chromosomal locations of the apoB and apoE structural genes in mice. The gene for apoB, designated Apob, resides in the proximal region of chromosome 12 linked to genes for ribosomal RNA and aryl hydrocarbon hydroxylase. The gene for apoE, designated Apoe, is located on chromosome 7, linked to genes for glucose phosphate isomerase and peptidase 4. Previously, we mapped the structural genes for apolipoproteins A-I and A-II to mouse chromosomes 9 and 1, respectively, and thus, the four loci encoding mammalian apolipoproteins have now been located in the mouse. These loci are homologous to the loci encoding apolipoproteins in humans as judged by the conservation of linked markers. A correlation was observed between a unique apoB allele and "responsiveness" to a high fat diet challenge. There were no obvious associations of apoB, apoE, or LDL/VLDL phenotypes or genotypes with diet-induced atherosclerosis among strains surveyed. These results clarify the organization and regulation of the genes for apoB and apoE, and they provide information about the naturally occurring polymorphisms affecting their expression.

Proteolytic processing and compartmentalization of the primary translation products of mammalian apolipoprotein mRNAs.

The steps involved in the initial assembly of apolipoproteins and lipids into supramolecular arrays (nascent lipoprotein particles) are largely unknown. Examination of the proteolytic processing and compartmentalization of the primary translation products of apolipoprotein mRNAs represents one approach to deciphering the molecular details of lipoprotein assembly. The structures of the primary translation products of seven mammalian apolipoprotein mRNAs has been determined in the past several years. The organization of apolipoprotein signal peptides is typical of eukaryotic prepeptides, although an unusual degree of sequence conservation is present among the signal segments of apo AI, AIV, and E. For those apolipoprotein sequences studied in detail, SRP-dependent cotranslational translocation and proteolytic processing appears to be highly efficient and results in sequestration of the processed protein within the lumen of the endoplasmic reticulum (ER). However the mechanism by which these lipid-binding proteins avoid arrest during their translocation through the lipid bilayer of the ER membrane remains obscure. The two principal human HDL apolipoproteins undergo novel extracellular post-translational proteolytic processing, which results in removal of nonhomologous propeptides. The proteases responsible for proapo AI and AII processing appear to be different. The processing of these proapolipoproteins provides a potential series of steps for regulating the ordered assembly of HDL constituents.