Cloning and expression of apolipoprotein B, the major protein of low and very low density lipoproteins.

Abstract

We report the cloning of cDNAs for rat liver apolipoprotein B (apo B) and the use of the cloned sequences to examine apo B expression at the level of mRNA in rat tissues. Fifteen putative apo B clones were identified by antibody screening of a rat liver cDNA library in the Xgtll expression vector. The identity of the clones was confirmed by immunological studies of the fusion protein products. All clones appear to contain sequences found only in apo B PI, the high molecular weight form of rat liver apo B. Blotting studies show that the clones hybridize to a single 20-kilobase liver mRNA species, sufficiently large to encode the entire apo B PI peptide, which is estimated to be 400 kDa in size. Apo B PI mRNA is abundant in liver and present in lower amounts in intestine but is absent in a variety of other tissues examined. This tissue distribution is consistent with that expected from studies on the in vivo synthesis of apo B. One clone, corresponding to a 240-base segment of the apo B PI mRNA, was sequenced and found to exhibit homology with a short region of rat apo E mRNA. Analysis of the secondary structure of the corresponding peptide did not show the preponderance of amphipathic a-helical structures characteristic of other apolipoproteins examined thus far.Apolipoprotein B (apo B) is a major protein component of mammalian very low density lipoproteins (VLDL) and chylomicrons and essentially the sole protein in low density lipoproteins (LDL). It is essential for the assembly/secretion of chylomicrons and VLDL, since these lipoproteins are absent in individuals with abetalipoproteinemia, a genetic disorder in which apo B is not produced (1). Apo B also functions as the ligand for the removal of LDL from the circulation by receptor-mediated uptake into a variety ofcells (2, 3).Despite intensive study, the structure of apo B has remained an enigma. It is among the largest peptides known, with estimates ranging from 250 to 550 kDa (1, 4-10), and is extremely hydrophobic as well as very sensitive to proteolytic degradation, and only recently has limited amino acid sequence information become available (11). Moreover, at least two major forms ofapo B are present in the circulation (8,9,(12)(13)(14)(15). In humans and most other mammals, the liver incorporates a 400-kDa species into VLDL, whereas the intestine incorporates a 210-kDa species into chylomicrons. However, the rat and mouse are exceptional in that their livers produce VLDL with comparable amounts ofboth these peptides (9,(13)(14)(15). We call these high and low molecular weight forms of apo B P(eptide)I and P(eptide)III, respectively; they are the B-100 and B-48 species in Kane's nomenclature (1,8,9). Although in the rat, PI is also accompanied by a slightly smaller component, PII, the two are very similar (9) and will be collectively referred to as PI. Peptide fingerprinting and immunological analysis suggest that PI encompasses a unique, PI-specific moiety joined to a second moiety which is identical or very similar to PIII (9,(16)(17)(18). Th...

Molecular cloning of partial cDNAs for rabbit liver apolipoprotein B and the regulation of its mRNA levels by dietary cholesterol.

“…Similar findings have been reported for rat (5) and human (7) intestine. Since the principal form of apoB synthesized and secreted by the intestine is apoB-48 (32), which has a molecular weight that is about one-half that of apoB-100, this raises the possibility that a separate mRNA exists for apoB-48, formed by differential splicing of the same apoB gene.…”

“…Based on an estimated molecular weight of 320,000 for rabbit apoB (32), a coding region of about 9 kb is needed for this protein. The 18-kb mRNA detected clearly fulfills this requirement and is consistent with the size of human and rat apoB mRNA (5)(6)(7)(8). The third line of evidence that supports the identification of XB-1 is that the 18-kb mRNA to which it hybridizes is present in high concentrations in liver and intestine relative to adrenal, brain, kidney, and spleen, consistent with the fact that liver and intestine are the only two tissues known to be actively involved in the synthesis and secretion of lipoproteins that contain apoB (1).…”

“…This in large part is due to its size, its insolubility when delipidated, its tendency to aggregate, and its susceptibility to oxidation. Some progress has recently been made, however, with the identification and sequencing of partial cDNA clones for human and rat apoB (5)(6)(7)(8)(9)(10).…”

Homozygous Familial Hypobetalipoproteinemia: Case Report

“…Apo B geni kromozom 2p24'te bulunur. 7 Apoprotein B eksikliği sonucu karaciğerden Apoprotein B içeren VLDL kolesterol sentezi azalırken barsaklarda da şilomikronlar oluşamamakta-dır. 8 Apo B-100 ve Apo B-48 adında iki subtipi vardır.…”

Human liver apolipoprotein B-100 cDNA: complete nucleic acid and derived amino acid sequence.

“…The heterogeneity in apoB-100 molecular size has been attributed to the propensity of delipidated apoB-100 to aggregate and to the reported sensitivity of apoB-100 to protease cleavage (3,4,7). We (8,9) and others (10)(11)(12)(13)(14)(15)(16) have reported the partial sequence of human and rat (17) liver apoB-100 cDNA. We now report the complete nucleotide sequence of apoB-100 mRNA and its derived amino acid sequence.…”