The role of ß-barrels 1 and 2 in the enzymatic activity of factor XIII A-subunit.

Essentials The roles of ß-barrels 1 and 2 in factor XIII (FXIII) are currently unknown. FXIII truncations lacking ß-barrel 2, both ß-barrels, or full length FXIII, were made. Removing ß-barrel 2 caused total loss of activity, removing both ß-barrels returned 30% activity. ß-barrel 2 is necessary for exposure of the active site cysteine during activation. SUMMARY: Background Factor XIII is composed of an activation peptide segment, a ß-sandwich domain, a catalytic core, and, finally, ß-barrels 1 and 2. FXIII is activated following cleavage of its A-subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis. Objectives To assess the functional roles of ß-barrels 1 and 2 in FXIII, we expressed and characterized the full-length FXIII A-subunit (FXIII-A) and variants truncated to residue 628 (truncated to ß-barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to ß-sandwich). Methods Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system. Results and Conclusions Circular dichroism spectroscopy and tryptophan fluorometry indicated that full-length FXIII-A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of ß-barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of ß-barrel 1 (TCC) restored enzymatic activity to ~ 30% of that of full-length FXIII-A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the ß-barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the ß-barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual ß-barrel domains in modulating access to the FXIII active site region.

Characterization of the rodent genes for arylacetamide deacetylase, a putative microsomal lipase, and evidence for transcriptional regulation.

In the current study, we have determined the cDNA and the genomic sequences of the arylacetamide deacetylase (AADA) gene in mice and rats. The AADA genes in the rat and mouse consist of five exons and have 2.4 kilobases of homologous promoter sequence upstream of the initiating ATG codon. AADA mRNA is expressed in hepatocytes, intestinal mucosal cells (probably enterocytes), the pancreas and also the adrenal gland. In mice, there is a diurnal rhythm in hepatic AADA mRNA concentration, with a maximum 10 h into the light (post-absorptive) phase. This diurnal regulation is attenuated in peroxisome proliferator-activated receptor alpha knockout mice. Intestinal but not hepatic AADA mRNA was increased following oral administration of the fibrate, Wy-14,643. The homology of AADA with hormone-sensitive lipase and the tissue distribution of AADA are consistent with the view that AADA plays a role in promoting the mobilization of lipids from intracellular stores and in the liver for assembling VLDL. This hypothesis is supported by parallel changes in AADA gene expression in animals with insulin-deficient diabetes and following treatment with orotic acid.

Microsomal malonyl-CoA-sensitive carnitine acyltransferase.

Liver microsomes contain two carnitine acyltransferase activities. One of these has properties closely corresponding to those of 88 kDa mitochondrial carnitine palmitoyltransferase-1 (CPT-1). Antisera against CPT-1 cross-react with an 88 kDa microsomal protein, suggesting that CPT-1 may be targeted to both microsomal and mitochondrial membranes. However, no experiments using cDNAs corresponding to CPT-1 involving in vitro translation with microsomes or involving in vivo COS-1 cell transfection provided any evidence to support this hypothesis.

Mobilisation of triacylglycerol stores.

Triacylglycerol (TAG) is an energy dense substance which is stored by several body tissues, principally adipose tissue and the liver. Utilisation of stored TAG as an energy source requires its mobilisation from these depots and transfer into the blood plasma. The means by which TAG is mobilised differs in adipose tissue and liver although the regulation of lipid metabolism in each of these organs is interdependent and synchronised in an integrated manner. This review deals principally with the mechanism of hepatic TAG mobilisation since this is a rapidly expanding area of research and may have important implications for the regulation of plasma very-low-density lipoprotein metabolism. TAG mobilisation plays an important role in fuel selection in non-hepatic tissues such as cardiac muscle and pancreatic islets and these aspects are also reviewed briefly. Finally, studies of certain rare inherited disorders of neutral lipid storage and mobilisation may provide useful information about the normal enzymology of TAG mobilisation in healthy tissues.

Metabolic characteristics of a human hepatoma cell line stably transfected with hormone-sensitive lipase.

Clones of HepG2 cells were selected that stably express the cDNA for hormone-sensitive lipase (HSL). When cells were cultured in the presence of labelled extracellular oleate, accumulation of labelled fatty acid as cellular triacylglycerol (TAG) was significantly lower in the transfectants compared with the wild-type cells. There was no change in the net rate of phospholipid (PL) synthesis. Culture of cells containing isotopically prelabelled TAG resulted in a greater net loss of TAG from the transfected cells than from the wild-type cells. The excess loss of labelled TAG was primarily due to an increased TAG fatty acid oxidation. Free fatty acid release into the medium was not increased in the transfectants, nor was the very low rate of lipoprotein lipid secretion. Also, there was no increased net trafficking of fatty acids from TAG into PLs. Changes in the 3H:14C ratio of TAG prelabelled with [3H]glycerol and [14C]oleate suggested that none of excess TAG fatty acid released in the transfected cells underwent intracellular re-esterification to TAG prior to oxidation. The results suggest that fatty acids mobilized by HSL are directed immediately into the oxidative pathway and are not available for biosynthetic processes. It appears likely, therefore, that intracellular TAG-derived fatty acids which enter the oxidative pathway exist in a different compartment from those that are directed towards synthesis.

Systematic expression of the complete coding sequence of apoB-100 does not reveal transmembrane determinants.

We have investigated the hypothesis that apolipoprotein B undergoes a regulated process of translocation into the endoplasmic reticulum (ER) which causes the protein to adopt a transmembrane configuration. Protein segments representing the complete coding sequence of apolipoprotein B were first expressed by in vitro translation of transcripts from seven overlapping transcripts. Two regions were identified (located at residue 2425 and between residues 4149 and 4348) that can undergo incomplete translocation into pancreatic microsomes. Ribosome pausing at these sites uncoupled translation from translocation, leading to the synthesis of large cytoplasmically oriented segments of protein. In contrast, when these two regions were expressed by transfection in cultured cells, transmembrane structures were not detected. Endogenous apolipoprotein B-100 synthesis in HepG2 cells generates a spectrum of nascent chains, indicating that ribosome pausing can also occur in intact cells. However, the cellular pause products were cotranslationally translocated. While endogenous apolipoprotein B-100 in HepG2 cells was fully translocated, discrete proteolytic fragments were generated from the amino terminus of the protein when proteases gained access to the lumen of permeabilized microsomes. These products were similar in size and sequence to apoliprotein B proteolytic fragments previously ascribed as the luminal domains of transmembrane apoB-100 molecules (Du, E. Z., Kurth, J., Wang, S. L., Humiston, P., and Davis, R. A. 1994. J. Biol. Chem. 269: 24169-24176).

Regulation of hepatic apolipoprotein-B-containing lipoprotein secretion.

The overall secretion of hepatic lipid as VLDL may be regulated by (i) changing the number of particles secreted as a result of altering apolipoprotein B output and (ii) changing the degree of lipidation of the particles with triacylglycerol so that their diameters vary over a threefold range. Substantial progress has been made in understanding the pathway of triacylglycerol incorporation into VLDL. Less is understood about the processes that commit apolipoprotein B either to secretion or to presecretory degradation, although both regulated translocation and an early lipidation step of the nascent particle with cholesterol or cholesterol ester have been implicated.

Microsomal triglyceride transfer protein activity remains unchanged in rat livers under conditions of altered very-low-density lipoprotein secretion.

Microsomal triglyceride transfer protein (MTP) is a heterodimeric protein consisting of a unique 97 kDa subunit and protein disulphide isomerase, that mediates the transfer of lipid between membranes and nascent lipoproteins. Mutations in the gene encoding the 97 kDa subunit of the protein cause the rare autosomal recessive disorder abetalipoproteinaemia. Recent findings in cultured cells indicate that the 5' promoter region contains an insulin-responsive element, suggesting that the gene is responsive to insulin regulation. In this study we examined two cases where very-low-density lipoprotein (VLDL) secretion is markedly reduced: the streptozotocin-diabetic rat and 10-day-old suckling rats. In both cases MTP activity was unaltered compared with that in control animals. Equal levels of MTP were also apparent in the livers of all groups of animals, as measured by immunoblotting with an anti-MTP antiserum. These findings indicate that factors other than MTP activity are responsible for the reduction in hepatic VLDL triglyceride secretion observed in the suckling and diabetic animals.

Studies on the translocation of the amino terminus of apolipoprotein B into the endoplasmic reticulum.

Apolipoprotein (apo) B is either co-translationally assembled into lipoproteins, or becomes associated with the membrane of the endoplasmic reticulum (ER) and is subsequently degraded. It has been proposed that apoB undergoes a novel process of translocation which generates cytoplasmically exposed apoB in the ER of hepatic and non-hepatic cells. Transmembrane forms of apoB can also be generated by in vitro translation (Chuck, S. L., and Lingappa, V. R. (1992) Cell 68, 9-21), which might explain the origin of untranslocated apoB in vivo. Here we have investigated a protocol which generates transmembrane forms of apoB during in vitro translation of truncated RNA transcripts. We observe that apoB can become transmembrane at sites of ribosome pausing and be held in this configuration by persistence of tRNA on the peptide chains. Ribosome pausing also occurs at these same sites in the absence of acceptor microsomes. Transmembrane topology can be generated at sites of ribosome pausing in a cytosolic protein, sea urchin cyclin when fused to a signal sequence. Mapping of the ribosome pause sites in apoB and in cyclin revealed no amino acid sequence homology. Chimeric constructs with engineered downstream glycosylation sites showed no evidence that ribosome pause sequences affect translocation of transcripts with termination codons. Transmembrane forms of apoB and cyclin were not generated during translocation into the ER in transfected COS cells.

Microsomal triglyceride transfer protein, the abetalipoproteinemia gene product, mediates the secretion of apolipoprotein B-containing lipoproteins from heterologous cells.

Apolipoprotein (apo) B is an obligatory component of triglyceride-rich lipoproteins. In the rare autosomal recessive disorder abetalipoproteinemia (ABL), no triglyceride-rich lipoproteins are secreted. Mutations in the gene encoding the 97-kDa subunit of a microsomal triglyceride transfer protein (MTP) cause ABL (Sharp, D., Blinderman, L., Combs, K. A., Klenzle, B., Ricci, B., Wager-Smith, K., Gil, C. M., Turck, C. W., Bouma, M. E., Rader, D. J., Aggerbeck, L. P., Gregg, R. E., Gordon, D. A., and Wetterau, J. R. (1993) Nature 365, 65-69; Shoulders, C. C., Brett, D. J., Bayliss, J. D., Narcisi, T. M., Jarmuz, A., Grantham, T. T., Leoni, P. R. D., Bhattacharya, S., Pease, R. J., Cullen, P. M., Levi, S., Byfield, P. G. H., Purkiss, P., and Scott, J. (1993) Hum. Mol. Genet. 2, 2109-2116). Here we have examined whether MTP is both necessary and sufficient to mediate the secretion of apoB-containing lipoproteins from cells that do not normally express either of these proteins. Carboxyl-terminal truncated forms of apoB, apoB17, and apoB41 on the centile system were expressed in COS-1 cells. ApoB17 was secreted whereas apoB41 was unable to traverse the secretory pathway. Cotransfection of apoB41 and MTP promoted the secretion of apoB41 as a buoyant lipoprotein particle with a modal density of 1.15 g/ml. When cotransfected COS-1 cells were cultured under conditions that increase the secretion of apoB100 from HepG2 cells, secretion of apoB41 was similarly increased. N-Acetyl-leucyl-leucyl-norleucinal (ALLN), a calpain I inhibitor, abolished intracellular degradation of apoB41 and increased secretion 2.5-fold. Oleate, a substrate for triglyceride synthesis, reduced degradation from 50 to 19% and increased secretion by 2.5-fold. The effects of ALLN and oleate were additive. We conclude that the secretion of apoB from COS-1 cells cotransfected with apoB and MTP is determined by the competitive processes of lipoprotein assembly and intracellular degradation in the endoplasmic reticulum and that MTP is the only tissue-specific component, other than apoB, required for the secretion of apoB-containing lipoproteins.

Abetalipoproteinemia is caused by defects of the gene encoding the 97 kDa subunit of a microsomal triglyceride transfer protein.

Abetalipoproteinemia is an inherited disorder of lipoprotein metabolism. Affected individuals produce virtually no circulating apolipoprotein B-containing lipoproteins (chylomicrons, very low density lipoprotein, low density lipoprotein and lipoprotein (a)). Malabsorption of the antioxidant vitamin E occurs, leading to spinocerebellar and retinal degeneration. Biochemical and genetic studies show that abetalipoproteinemia is not a defect of lipid biosynthesis or of the apolipoprotein B gene. Instead a microsomal triglyceride transfer protein, which exists as a complex with protein disulphide isomerase in the endoplasmic reticulum, has been implicated. We have cloned and sequenced the human cDNA encoding microsomal triglyceride transfer protein. The predicted amino acid sequence shows extensive homology to vitellogenin, the precursor of the lipovitellin complex, which has been shown by X-ray crystallography to contain a large lipid storage cavity. Microsomal triglyceride transfer protein is expressed in ovary, testis and kidney, in addition to liver and small intestine. A homozygous mutation that disrupts splicing has been identified in affected siblings with classical abetalipoproteinemia. These results elucidate a key process in the packaging of apolipoprotein B with lipid, and should increase our understanding of the processes regulating the production of atherogenic lipoproteins.

Oleate-mediated stimulation of apolipoprotein B secretion from rat hepatoma cells. A function of the ability of apolipoprotein B to direct lipoprotein assembly and escape presecretory degradation.

Physiological concentrations of oleate stimulate apolipoprotein (apo) B-containing lipoprotein secretion from HepG2 cells without increasing apoB mRNA levels. The purpose of this study was to determine whether oleate acts by increasing translation of apoB mRNA or through posttranslational effects on the apoB protein. To address the mechanism of oleate-stimulated secretion of apoB, a series of carboxyl terminally truncated apoB constructs was made. Each contained the SV40 early promoter, the apoB 5'-untranslated region, and SV40 polyadenylation signals. Any difference in the response to oleate between endogenous apoB and the proteins encoded by the constructs or between the constructs themselves should thus depend on the protein sequence. Stable transformants were established for each of the constructs in the rat hepatoma cell line McArdle-RH7777. The effect of oleate on secretion of the apoB protein products was determined by labeling with [35S]methionine, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carboxyl-terminal truncation of apoB41 resulted in a loss of the ability of apoB secretion to respond to oleate. Ultracentrifugation of secreted proteins on continuous CsCl gradients from 1.0-1.4 g/ml revealed that this correlated with a decrease in the ability of apoB to be recovered as a buoyant lipoprotein particle. Addition of oleate decreased the densities at which the short forms of apoB secreted as lipoproteins were recovered. Pulse-chase analysis of the secretion of apoB100 and of the truncated proteins revealed that they all underwent rapid posttranslational intracellular degradation. We conclude that oleate has no effect on the translation of apoB mRNA but promotes the secretion of apoB-containing lipoproteins by reducing presecretory degradation of those forms of apoB that can produce buoyant lipoproteins.

Biochemical analysis of the mouse mammary tumor virus long terminal repeat product. Evidence for the molecular structure of an endogenous superantigen.

Recent reports have shown that both exogenous and endogenous mouse mammary tumor viruses (MMTV) can encode superantigens. Transfection and transgenic studies have identified the open reading frame (ORF) present in the 3' long terminal repeat (LTR) as encoding superantigen function. In this study, we have used an in vitro translation system in an attempt to characterize the molecular nature of the protein encoded by the 3' ORF of Mtv-8. Using various constructs encoding full-length and truncated versions of the ORF product, we report that the hydrophobic region close to the amino terminus of the 36-kDa protein can function as a transmembrane domain. Protease digestion experiments also demonstrate that the protein has a type-II transmembrane conformation with an extra-cytoplasmic carboxy terminus. Since this hydrophobic region is conserved between all known MMTV, we speculate that LTR ORF, including those proposed to encode the minor lymphocyte stimulatory antigens, are also capable of encoding type-II transmembrane glycoproteins. The polymorphism between MMTV LTR ORF products, which correlates with deletion phenotypes, is predominantly in the carboxy-terminal extracellular region, consistent with a major role in interaction with the T cell receptor.

Cotranslocational insertion of apolipoprotein B into the inner leaflet of the endoplasmic reticulum.

Apolipoprotein (apo) B100 is required for the distribution of hepatic triglyceride to peripheral tissues as very-low-density lipoproteins. The translocation of apo B100 into the endoplasmic reticulum (ER) and its subsequent assembly into lipoprotein particles is of particular interest as the protein is both very large (relative molecular mass 512,000) and insoluble in water. It has been proposed that apo B translocation occurs in discrete stages and is completed post-translationally. Several sites of arrest of translocation were reported to be present in apo B15 (the N-terminal 15% of the protein). We have re-examined this question by in vitro translation coupled with translocation into microsomes, and find no evidence for transmembrane segments in truncated apo B proteins. Translocated apo B17 is strongly associated with the membrane of the ER, being only partially releasable with alkaline carbonate, and remaining bound to the microsomes following disruption with saponin. The efficient binding of short segments of apo B, despite the absence of transmembrane domains, suggests that apo B is cotranslationally inserted into the inner leaflet of the ER. This will obviate problems caused by the size and insolubility of apo B100, because the growing hydrophobic protein chains will never exist in a lipid-free form during translocation. From the inner leaflet, apo B in association with membrane-derived lipid can bud into the lumen of the ER to form nascent lipoprotein particles.

Carboxyl-terminal truncation of apolipoprotein B results in gradual loss of the ability to form buoyant lipoproteins in cultured human and rat liver cell lines.

Apolipoprotein B has an obligatory role in the production of chylomicrons, VLDL, and LDL. Familial hypobetalipoproteinemia is a codominant disorder characterized by reduced levels of apo B containing lipoproteins in plasma. We have previously described mutations of the apo B gene in persons with hypobetalipoproteinemia that predict truncated forms of apo B designated apo B29 (1305 amino acid residues) and apo B39 (1799 residues). Apo B39 was present in the VLDL and LDL fractions of plasma, but apo B29 was not detected in the lipoprotein or infranatant fractions of plasma. Here we have investigated the regions of apo B necessary for apo B containing lipoprotein secretion by expression of constructs designed to express truncated forms of apo B. Apo B13 (583 residues), apo B17 (784 residues), apo B23 (1084 residues), apo B29 (1306 residues), and apo B41 (1880 residues) were transiently expressed in HepG2 cells, and apo B23 and apo B41 were stably expressed in McArdle 7777 cells. Lipoprotein (d less than 1.25 g/mL) and infranatant (d greater than 1.25 g/mL) fractions of conditioned medium were analyzed by immunoprecipitation and SDS-PAGE. The distribution between lipoprotein and infranatant fractions varied: apo B41 was found solely in the lipoprotein fraction; apo B29, apo B23, and apo B17 were present in both fractions, but with stepwise truncation, progressively more apo B was recovered in the infranatant; apo B13 was only in the infranatant. These results demonstrate that deletion from the carboxyl terminal of apo B41 results in a gradual loss of the ability of the truncated proteins to form buoyant lipoprotein particles.

Conformation of apolipoprotein B-100 in the low density lipoproteins of tangier disease. Identification of localized conformational response to triglyceride content.

The low density lipoproteins (LDL) from patients with Tangier disease are enriched in triglycerides, 27% of LDL mass versus 7% for normal LDL. To study whether this unique LDL core lipid composition affects the surface disposition of apolipoprotein (apo) B-100, we analyzed the LDL by protease digestion and in competitive radioimmunoassays. Limited proteolytic digestion of Tangier LDL by Staphylococcus aureus V8 protease generated a prominent fragment of 120 kDa (cleavage site at residue 1076), which was not visible in similarly digested normal LDL. In competitive radioimmunoassay, Tangier LDL bound weakly to the apoB-specific monoclonal antibody MB20, compared with control LDL. We localized the MB20 epitope between residues 1031 and 1084 of apoB-100, probably very near residue 1076. DNA sequencing of exon 21 of apoB genomic clones (coding for residues 1014-1084) from a Tangier patient revealed no difference from the normal DNA sequence, thus eliminating a protein polymorphism as a basis for the altered protease sensitivity and antibody binding. When the triglyceride contents of Tangier LDL were reduced to 10% of mass by incubation with normal high density lipoproteins, production of the 120-kDa fragment by proteolysis decreased and MB20 binding increased in affinity, implying a change toward normal conformation of apoB-100. Thus, using two independent techniques, proteolytic digestion and binding of monoclonal antibodies, we have demonstrated an alternative conformation of apoB-100 in the vicinity of residue 1076, which reflects the content of triglycerides in the LDL particle.

Use of bacterial expression cloning to localize the epitopes for a series of monoclonal antibodies against apolipoprotein B100.

Bacterial expression of apolipoprotein (apo) B cDNA constructs has been used to map a series of monoclonal antibodies (mAbs) to apoB by immunoblotting. In some cases assignments have been confirmed and refined by (i) semipurification of expressed protein, CNBr digestion, and assignment of the immunoreactive fragments; (ii) controlled digestion of the cDNA with the exonuclease Bal31 and bacterial expression of the truncated proteins that result; or (iii) expression of specific segments of cDNA amplified by the polymerase chain reaction. Forty mAbs were mapped to a minimum of 17 separate determinants on apoB. Tryptic fragments have been used to confirm the epitope assignments. In addition, this approach in conjunction with immunoassay, enables some deductions to be made about the trypsin-accessible regions in low density lipoprotein (LDL). The cleavage pattern obtained predicts retention of structure in the cysteine-rich domain of the amino terminus and also in the LDL receptor binding region. Trypsinized LDL was shown to bind to the LDL receptor by an authentic process, using monoclonal antibodies as competing ligands. In conjunction with the previous paper (Milne, R. W., Theolis, R., Maurice, R., Pease, R. J., Weech, P. K., Rassart, E., Fruchart, J.-C., Scott, J., and Marcel, Y. L. (1989) J. Biol. Chem. 265, 19754-19760) the mapped mAbs have been used to define the receptor-binding domain of apoB100 in LDL.

The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B.

Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.

Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia.

Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here we describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant, apo-B(His1795----Met-Trp-Leu-Val-Thr-Term) is predicted to be 1799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant, apo-B(Arg1306----Term), is caused by mutation of a CpG dinucleotide in arginine codon 1306 converting it to a stop codon and predicting a protein of 1305 residues. The product of this allele could not be detected in the circulation. The differences in size and behaviour of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins.

Apolipoprotein B: a novel mechanism for deriving two proteins from one gene.

Evidence from antibody and peptide mapping, protein sequencing and amino acid composition studies suggests that intestinal apo-B48 is colinear with the amino terminal half of hepatic apo-B100. To investigate the mechanism of apo-B48 production we examined cDNA clones from human and rabbit small intestine. All clones contained a single C----T base difference from the hepatic sequence resulting in a translational stop at codon 2153. Amplification by the polymerase chain reaction of cDNA from human and rabbit small intestine, rabbit liver and the human hepatoma cell line HepG2 showed that the stop codon was only present in intestinal mRNA. Enterocyte genomic DNA did not contain the stop codon. We suggest that a co- or post-transcriptional C----U change may result in the production of apo-B48, which represents the amino terminal 2152 amino acids of apo-B100. This is the first example of tissue specific modification of a single mRNA nucleotide resulting in two different proteins from the same primary transcript.