Biomedical subjects

J I Gordon

Publications and source records attributed to J I Gordon.

At least 253 records · Page 14Linked to original sources

PubMed1986

Tissue-specific expression and developmental regulation of the rat apolipoprotein B gene.

Expression of the apolipoprotein B (apoB) gene was examined in a variety of fetal, neonatal, and adult rat tissues by probing RNA blots with a cloned rat apoB cDNA. Among 10 adult male tissues surveyed, small intestine had the highest concentration of apoB mRNA. Its abundance in liver and adrenal gland was 40% and 0.5%, respectively, of that in small bowel, while none was detected in colon, kidney, testes, spleen, lung, heart, or brain. ApoB mRNA is as abundant in 18-day fetal liver as at any subsequent period of hepatic development. In contrast, the concentration of apoB mRNA remains low in fetal intestine until the last (21st) day of gestation, when it increases sharply to levels that are several-fold higher than in the liver. ApoB mRNA levels in fetal membranes harvested during this late gestational period were 10 times greater than in fetal liver. Since the major lipoprotein species in 19-day fetal plasma is low density lipoprotein, these observations suggest that fetal liver, and particularly its functional homologue, the yolk sac, are the principal sites of fetal lipoprotein synthesis at this stage of development. A 20-fold increase in placental apoB mRNA concentrations during the last 48 hr of pregnancy (to a level that is 50% of that encountered in fetal membrane RNA) suggests a specific role for this organ in maternal-fetal lipid transport immediately prior to parturition. Pulse-labeling experiments using 21-day fetal tissue slices showed that the liver synthesizes both apoB-100 (B-PI) and apoB-48 (B-PIII) albeit in somewhat different ratios than the adult organ. Fetal intestine produces almost exclusively the smaller apoB species, while fetal membranes and placenta synthesize only the larger peptide. The postnatal pattern of apoB mRNA accumulation is similar in liver and intestine. Profound decreases were observed during the late suckling and weaning periods, followed by an increase to adult levels. These final concentrations were similar to those encountered at birth. Analysis of these developmental changes offers an opportunity to generate testable hypotheses about the factors that modulate apoB synthesis.

Age Factors↗

PubMed1986

Developmental regulation of a gene that encodes a cysteine-rich intestinal protein and maps near the murine immunoglobulin heavy chain locus.

Mouse and rat small intestinal cDNA libraries were screened for recombinants derived from mRNAs whose concentration changed during the transition from suckling to weaning. cDNAs transcribed from a 570-nucleotide-long mRNA were isolated. Dot blot hybridization analyses of RNA recovered at various stages of rat gastrointestinal ontogeny indicated that the concentration of this mRNA begins to increase during the mid-suckling period, reaching a peak during weaning. There is considerable variation in the relative amount of this mRNA in adult tissues, with highest levels encountered in the rat small intestine and colon. Its concentration in duodenum, jejunum, and ileum is approximately the same. It is more concentrated in villi than in crypts. The rat mRNA encodes a 77 amino acid, 8.55-kDa polypeptide that has seven cysteine residues. This cysteine-rich intestinal protein (named CRIP) has two internal repeated sequence blocks. Computer-assisted comparisons of CRIP to proteins of known function disclosed that it is homologous to certain ferredoxins. Southern blot analyses revealed that sequences homologous to the rat gene are present in sea squirt, fish, bird, and human DNA, indicating that this gene is highly conserved and that related proteins may be present in many if not all vertebrates. Recombinant inbred mouse strains were utilized to show that the CRIP gene is closely linked to the immunoglobulin heavy chain constant region locus, Igh-c, on chromosome 12. CRIP mRNA is a molecular marker for the suckling-to-weaning transition of rodent intestinal development. The cloned cDNA may be a useful probe for identifying factors that regulate intestinal development during this period.

Animals↗

PubMed1986

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.

Animals↗

PubMed1986

On computer-assisted analysis of biological sequences: proline punctuation, consensus sequences, and apolipoprotein repeats.

During the past several years, the use of computer programs in the analysis of protein and DNA sequences has become commonplace. In all but the simplest procedures, the ability to critically review the results obtained with computer methods requires a basic knowledge of the algorithms employed (and the assumptions upon which they are based), an awareness of the capabilities and limitations of the particular program that implements an algorithm, and some familiarity with probability and statistics. We describe a number of computer methods that have been applied to the analysis of apolipoprotein sequences. We discuss the suitability of these methods for particular problems, how the choice of initial "parameters" can affect the results, and what the results can tell us about protein or gene sequences. We also identify some outstanding problems of apolipoprotein sequence analysis where further work is needed.

Amino Acid Sequence↗

PubMed1985

Extracellular processing of proapolipoprotein A-II in Hep G2 cell cultures is mediated by a 54-kDa protease immunologically related to cathepsin B.

Apolipoprotein A-II is the second most abundant polypeptide found in human plasma high density lipoprotein particles. The primary translation product of human apo-A-II mRNA is a prepropolypeptide. We have previously reported (Gordon, J. I., Sims, H. F., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1984) J. Biol. Chem. 259, 15556-15563) that the prosegment of apo-A-II was removed following export from a human hepatoma cell line (Hep G2). This represented a novel processing compartment for prosegments terminating with paired basic residues and differed from the processing of proalbumin which occurred with high efficiency prior to export from these cells. We have now characterized the enzyme responsible for this extracellular cleavage. The proapo-A-II converting activity is blocked by the thiol protease inhibitors antipain, E-64, leupeptin, and Ala-Lys-Arg chloromethyl ketone. Incubation of 125I-iodotyrosylated Ala-Lys-Arg chloromethyl ketone with serum-free media harvested from cell cultures over a 12-h period revealed a time-dependent accumulation of a 54-kDa protease. Although small quantities of the 54-kDa protease were detected in cell lysates, the major intracellular sequences labeled by the affinity probe had masses of 31.5 and 6 kDa. The 54-kDa extracellular, as well as 31.5- and 6-kDa intracellular, species were all immunoprecipitated by monospecific anti-human liver cathepsin B IgG. Addition of this antibody to media inhibited extracellular conversion of proapo-A-II to the mature protein. Based on these observations, we conclude that a "pro" cathepsin B-like protease exported by Hep G2 cells is responsible for proapo-A-II prosegment removal. It appears that cathepsin B-like proteases exhibit a complex pattern of segregation within the secretory pathway and that larger molecular weight forms of cathepsin B-like proteases are capable of accurately processing propolypeptides.

Apolipoprotein A-II↗

PubMed1985

Analyzing the structures, functions and evolution of two abundant gastrointestinal fatty acid binding proteins with recombinant DNA and computational techniques.

The structures of intestinal and liver fatty acid binding proteins (FABPs) have been determined from an analysis of the nucleotide sequences of cloned cDNAs. The primary translation product of intestinal FABP mRNA contains 132 residues (Mr = 15 124). Liver FABP mRNA encodes a 127 amino acid polypeptide (Mr = 14 273). In vitro co-translational cleavage and translocation assays showed that neither sequence has a cleavable signal peptide or signal peptide equivalent - suggesting that the FABPs do not enter the secretory apparatus but rather are targeted to the cytoplasm. A variety of computational techniques were used to compare the two FABP sequences. The results indicate that liver and intestinal FABP are paralogous homologues. A superfamily of proteins was defined which includes the FABPs, the cellular retinol and retinoic acid binding proteins, the P2 protein of peripheral nerve myelin, and a polypeptide known as 422 whose synthesis is induced during differentiation of 3T3-L1 cells to adipocytes. No sequence homologies were noted between any of these small molecular weight cytosolic proteins and nonspecific lipid transfer protein (sterol carrier protein 2), phosphatidylcholine transfer protein, serum albumin or apolipoprotein AI. The FABPs may have structural features responsible for lipid-protein interactions that are not present in these non-homologous sequences. The distribution of intestinal and liver FABP mRNAs in adult rat tissues and the changes in FABP gene expression which occur during gastrointestinal development support the notion that these proteins are involved in fatty acid uptake, transport and/or compartmentalization. However, differences in tissue distribution and periods of non-coordinate expression during gastrointestinal ontogeny suggest that the two FABPs have distinct functions. The relationship between intestinal and liver FABPs and similar sized cytosolic FABPs isolated from brain, skeletal and cardiac muscle remains unclear. Recombinant DNA techniques combined with comparative sequence analyses offer a useful approach for defining unique as well as general structure-function relationships in this group of fatty acid binding proteins.

Amino Acid Sequence↗

PubMed1985

Human liver fatty acid binding protein. Isolation of a full length cDNA and comparative sequence analyses of orthologous and paralogous proteins.

We have determined the primary structure of human liver fatty acid binding protein from an analysis of a full length cDNA. This 127-residue 14,178-Da protein exhibits a high degree of sequence conservation when compared to its orthologous homologue, rat liver fatty acid binding protein. It appears likely that this polypeptide arose from two intragenic duplication events. Using a variety of computational techniques, we were unable to find any evidence of amphipathic alpha helical domains in this protein nor any sequence similarities to apolipoproteins and serum albumins. A family of paralogous proteins was defined, whose members share a remarkable degree of sequence homology with share a remarkable degree of sequence homology with human liver fatty acid binding protein. These include rat intestinal fatty acid binding protein, the cellular the P2 protein of myelin. It appears that the small cytosolic fatty acid binding proteins have evolved structural features necessary for lipid-protein interaction which are different from those present in some familiar and better studied extracellular sequences.

Amino Acid Sequence↗

PubMed1985

Tissue specific expression and developmental regulation of two genes coding for rat fatty acid binding proteins.

We have examined the tissue distribution and developmental regulation of two low molecular weight cytosolic fatty acid binding proteins. Based on their initial site of isolation, they have been referred to as liver and intestinal fatty acid binding proteins (FABP). Cloned cDNAs were used to probe blots of RNAs extracted from a wide variety of adult rat tissues as well as small intestine and liver RNA obtained from fetal, suckling, and weaning animals. The highest concentrations of "liver" FABP mRNA were found in small intestine and liver. "Intestinal" FABP mRNA is most abundant in small bowel RNA while only trace amounts were encountered in liver. Both mRNAs were detectable in stomach, colon, pancreas, spleen, lung, heart, testes, adrenal, and brain RNA at 1-8% the concentrations observed in small intestine. Accumulation of both mRNAs in the small intestinal epithelium increases during development. The mRNAs are first detectable between the 19th and 21st day of gestation. They undergo a coordinated 3-4-fold increase in concentration within the first 24 h after birth. Thereafter, gut levels of intestinal FABP mRNA remain constant during the suckling period while liver FABP mRNA increases an additional 2-fold. Liver FABP mRNA levels are also induced in hepatocytes during the first postnatal day but subsequently do not change during the suckling and weaning phase, despite marked alterations in hepatic fatty acid metabolism. These observations support the concept that the major role of these proteins is to facilitate the entry of lipids into cells and/or their subsequent intracellular transport and compartmentalization. The data also raise questions about the identity of extragastrointestinal FABPs.

Aging↗

PubMed1985

Regulation of the biosynthesis of two distinct fatty acid-binding proteins in rat liver and intestine. Influences of sex difference and of clofibrate.

Two distinct fatty acid-binding proteins (FABPs) have been identified in rat intestine, gFABP (15,063 Da) which is confined to intestinal epithelium and hFABP (14,184 Da) which is found in both liver and intestine. We have examined the influence of sex difference and the effect of clofibrate, both of which affect cellular fatty acid metabolism and hFABP levels, on the concentration, and mRNA levels of both hepatic and intestinal FABPs. In the liver, hFABP concentration was approximately 2-fold greater in females and in clofibrate-treated males than in untreated male rats. These differences were not accompanied by changes in the fractional turnover of the polypeptide but rather by parallel increases in hFABP mRNA. In the intestine, the two FABPs exhibited different regulatory responses. Intestinal hFABP turnover was 33% greater in females than in males, whereas mRNA concentration was 50% greater. Thus, unlike hFABP in liver, there was no sex-related difference in the steady-state level of hFABP in intestine. However, clofibrate treatment, similar to its effects in the liver, doubled intestinal hFABP protein and mRNA concentration. In contrast to hFABP, neither gFABP protein nor mRNA concentration were sex dependent, whereas clofibrate produced only a modest increase in gFABP concentration without significantly changing gFABP mRNA levels. The results indicate that the influence of sex difference and the effect of clofibrate on hepatic fatty acid metabolism are both associated with changes in hFABP synthesis mediated pretranslationally. The differential response of hFABP and gFABP in intestine suggests that these proteins play distinct roles in the cellular metabolism of fatty acids.

Animals↗

PubMed1985

Expression of rat apolipoprotein A-IV and A-I genes: mRNA induction during development and in response to glucocorticoids and insulin.

Rat apolipoproteins (apo-) A-IV and A-I share many structural similarities, the most notable of which is a domain of repeated docosapeptides with amphipathic helical potential. Although the genes for apo-A-IV and apo-A-I probably diverged from a common ancestor, these proteins seem to have developed different functions in their evolution. In the present study, cloned cDNAs were used to characterize the expression of apo-A-IV and apo-A-I mRNAs in a wide variety of adult rat tissues, as well as in small intestine and liver obtained from fetal, suckling, and weanling animals; comparisons were made to the expression of apo-E mRNA. The apo-A-IV and apo-A-I mRNAs were most abundant in adult small intestine and liver, with trace amounts detected in other tissues. Substantial amounts of these mRNAs were detected in the yolk sac, suggesting that this fetal tissue plays an important role in lipid metabolism during gestation. Noncoordinate accumulation of apo-A-IV and apo-A-I mRNAs was observed within and between the liver and small intestine during neonatal development. The apo-A-IV mRNA levels in the developing small intestine and liver appeared to correlate with their triglyceride secretion rates, suggesting that this protein plays an important role in the metabolism of triglyceride-rich lipoproteins. When dexamethasone (0.1 microM), insulin (0.01 microM), or insulin and dexamethasone together were incubated with primary cultures of nonproliferating adult rat hepatocytes, apo-A-IV mRNA levels were 4-, 7-, and 11-fold higher, respectively, than in non-hormone-treated control hepatocytes. Hormone administration resulted in a 2-fold greater amount of apo-A-I mRNA in each case, with no significant change in the level of apo-E mRNA. The overall results suggest that these structurally related apolipoproteins are regulated in substantially different ways.

Animals↗

PubMed1985

Comparative analysis of repeated sequences in rat apolipoproteins A-I, A-IV, and E.

To understand the structural, functional, and evolutionary relationships among the principal protein components of rat high density lipoprotein particles, we undertook a systematic comparative analysis of the primary structures of apolipoproteins (apo)-A-I, -A-IV, and -E. Human apo-A-I and rat apo-A-IV have been shown previously to contain repeated sequences that presumably arose by intragenic duplication of 11- or 22-amino acid amphipathic segments. For apo-A-I, these segments are thought to be the structures responsible for lipid binding and activation of lecithin:cholesterol acyltransferase. From an analysis of the sequence of a full-length cDNA clone, rat apo-A-I is shown to contain eight tandem repetitions of a 22-amino acid segment. However, compared with human apo-A-I, the rat protein has undergone three deletions, two of which involve multiple amino acids in the repeated sequence domain. This disruption of the periodic structure of the protein raises the possibility of species-specific variation in the ability of rat apo-A-I to interact with high density lipoproteins and activate lecithin:cholesterol acyltransferase. Statistical analysis of the structure and organization of repeated sequences in apo-A-I, -A-IV, and -E demonstrates that all three proteins are paralogous members of a dispersed gene family. Despite overall similarity in sequence organization, different portions of these sequences have evolved at different rates. Diversification of a duplicated ancestral sequence has resulted in three lipid-binding proteins with distinct and shared functions.

Amino Acid Sequence↗

PubMed1984

Human proapolipoprotein A-II is cleaved following secretion from Hep G2 cells by a thiol protease.

The two principal high-density lipoprotein apolipoproteins A-I and A-II are both initially synthesized as preproproteins. The prosegment of apo-A-I is unusual: it ends with paired glutamine residues and is removed extracellularly. The apo-A-II prosegment resembles the propeptides of prohormones and proalbumin: it ends with paired basic amino acids. We have studied the processing of proapo-A-II in a human hepatoma cell line (Hep G2) which is known to accurately and efficiently remove the prosegment from proalbumin prior to secretion. Pulse-chase experiments were performed in order to determine if the apo-A-II prosegment is removed prior to or after secretion. Apo-A-II was purified from cell lysates and media at various times during the chase and subjected to automated sequential Edman degradation. The results indicate that proteolytic processing of proapo-A-II is largely an extracellular event. These cells secrete the protease responsible for prosegment removal. The converting activity present in media is not blocked by serine protease inhibitors (phenylmethanesulfonyl fluoride, aprotinin, and furoyl saccharin) or by a metalloprotease inhibitor (o-phenanthroline). It is inhibited by the thiol protease reagents p-chloromercuribenezene-sulfonic acid and leupeptin. Prosegment removal changes the pI of the dominant apo-A-II isoform from 6.61 to 4.95. The presence of the propeptide does not prevent specific in vitro recombination of apo-A-II with high-density lipoprotein3 particles present in normolipemic serum. Extracellular processing after a single basic amino acid has been described for a variety of precursor proteins. Extracellular cleavage of the apo-A-II propeptide after paired COOH-terminal basic residues represents a novel processing pathway.

Amino Acid Sequence↗

PubMed1984

The enzyme that cleaves apolipoprotein A-II upon in vitro incubation of human plasma high-density lipoprotein-3 with blood polymorphonuclear cells is an elastase.

The proteolytic activity directed against apolipoprotein A-II (apo-A-II) which is released from human blood polymorphonuclear cells (PMN) when they are incubated with human plasma high-density lipoprotein-3 (HDL3) was studied to assess the properties and site specificity of the enzyme. When 125I-apo-A-II-labeled HDL3 was incubated with the PMN protease at 37 degrees C, a complete cleavage of apo-A-II was observed which paralleled the formation of bands of approximately 11,000 and 7,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 7,000-dalton component had the following N-terminal sequence: NH2-Thr-Asp-Tyr-Gly-Lys-Asp-Leu-Met-Glu-Lys. This corresponds to residues 19 through 28 of the intact apo-A-II monomer. Methoxysuccinyl (MeO-Suc)-Ala-Ala-Pro-Val-chloromethylketone-(CH2Cl) caused a 90% inhibition of apo-A-II hydrolysis at the highest concentration tested (6 X 10(-4)M). Besides apo-A-II, the PMN enzyme also hydrolyzed a synthetic substrate, MeO-Suc-Ala-Ala-Pro-Val-4-nitroanilide and its 4-methylcoumaryl-7-amide analogue. The protease appeared to have a mass of 28,000 daltons as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]diisopropylfluorophosphate-labeled PMN enzyme. That the PMN enzyme which cleaves apo-A-II is an elastase was derived from the following criteria: 1) cleavage at the Val-X bond in apo-A-II and in the two synthetic substrates studied; 2) prevention of the cleavage by MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, a known specific elastase inhibitor; and 3) a mass comparable to that reported for a pure PMN elastase. These studies establish that apolipoproteins can be suitable substrates for enzymes of the elastase family.

Apolipoprotein A-II↗

PubMed1984

Expression of a mammalian fatty acid-binding protein in Escherichia coli.

Rat liver fatty acid-binding protein (FABP) is a 14,200-Da polypeptide that is abundantly represented in the cytosol of liver and small intestinal epithelial cells. It may play an important role in the intracellular transport and metabolism of fatty acids. We have recently determined its sequence from an analysis of cloned cDNAs (Gordon, J.I., Alpers, D.H., Ockner, R. K., and Strauss, A.W. (1983) J. Biol. Chem. 258, 3356-3363). With the availability of full-length cloned FABP cDNAs, genetic manipulation of the coding region introduced into an appropriate expression vector could provide a powerful tool for analyzing the molecular details of fatty acyl-protein interaction. We, therefore, have inserted the coding sequence of rat FABP cDNA into a prokaryotic expression vector. Synthetic oligodeoxynucleotides were used to "properly" space the initiator methionine residue downstream from a Shine-Dalgarno ribosome-binding site. FABP transcription in the chimeric plasmid was under the direction of the leftward promotor of phage lambda. Promotor activity in turn was regulated by a thermolabile repressor specified by a defective lambda prophage contained in the host chromosome. When grown at permissive temperatures, rat liver FABP represented approximately 0.8% of radiolabeled soluble bacterial proteins. Edman degradation of FABP purified from Escherichia coli lysates indicated that it was intact. However, its NH2 terminus was not acetylated as it is in mammalian tissues. A frameshift mutation was introduced in vitro into the coding region of FABP cDNA. The effects of this on fatty acyl-protein interaction were examined using a solid phase (oleic acid-Sepharose) binding assay. Amino acid sequence rearrangements in the COOH-terminal region of rat liver FABP had a significant effect on its ability to bind fatty acids as well as on its stability in bacteria.

Amino Acid Sequence↗

PubMed1984

Biosynthesis and compartmentalization of rat-intestinal vitamin-D-dependent calcium-binding protein.

We have purified the primary translation product of rat intestinal vitamin-D-dependent calcium-binding protein mRNA from wheat germ and ascites cell-free systems. We show that calcium-binding protein is neither synthesized as a larger percursor nor likely to be exported from the intestinal epithelium. Our conclusions are based on the following observations. (1) The primary translation product, NH2-terminally labeled with formyl[35S]methionine, comigrates with the mature cytoplasmic protein during electrophoresis through denaturing gels. (2) It does not possess a cleavable signal peptide sequence or internal signal equivalent as judged by co- and post-translational cleavage assays in vitro. (3) The NH2 terminus of the cell-free product is acetylated. (4) Comparison of the NH2-terminal amino acid sequences of the primary translation product and cyanogen bromide peptides obtained from the blocked, purified cytoplasmic protein. The kinetics of calcium-binding protein mRNA accumulation and decay in rachitic intestinal epithelium after primary and secondary stimulation with 1,25-dihydroxycholecalciferol (calcitriol) were studied using the cell-free translation system. The results are reminiscent of other steroid-hormone-inducible systems. Both the rate of mRNA accumulation and the peak response were greater after secondary stimulation.

Amino Acid Sequence↗

PubMed1984

Biosynthesis of apolipoprotein C-III in rat liver and small intestinal mucosa.

We have examined the biosynthesis of rat apolipoprotein C-III in the small intestine and liver. The primary translation product of its mRNA was recovered from wheat germ and ascites cell-free systems. Comparison of its NH2-terminal sequence with the NH2 terminus of plasma high density lipoprotein-associated apolipoprotein C-III showed that apo-C-III was initially synthesized as a preprotein with a 20 amino acid long NH2-terminal extension: Met-X-X-X-Met-Leu-Leu-X-X-Ala-Leu-X-Ala-Leu-Leu-Ala-X-Ala-X-Ala. Co-translational cleavage of the cell-free translation product by signal peptidase generated a polypeptide with the same NH2 terminus as the mature protein (X-Glu-X-Glu-Gly-Ser-Leu-Leu-Leu-Gly-Ser-Met). Therefore, this apolipoprotein does not undergo post-translational proteolytic processing like two other high density lipoprotein-affiliated proteins, proapo-A-I and proapo-A-II. The mRNA encoding apolipoprotein C-III comprises 0.4% of the translatable RNA species in adult rat liver and 0.14% of the translatable RNA species in small intestinal epithelium. Acute fat feeding with a triglyceride meal resulted in a 2-fold increase in intestinal preapo-C-III mRNA accumulation but no change in the levels of preproapo-A-I mRNA. Thus, the acute response of the apo-A-I and C-III genes to triacylglycerol absorption differs.

Amino Acid Sequence↗

PubMed1984

Biosynthesis of human preapolipoprotein A-IV.

The primary translation product of human intestinal apolipoprotein A-IV mRNA was purified from ascites and wheat germ cell-free systems. Comparison of its NH2-terminal sequence with mature, chylomicron-associated apo-A-IV revealed that apo-A-IV was initially synthesized with a 20-amino acid long NH2-terminal extension: Met-X-Leu-X-Ala-Val-Val-Leu-X-Leu-Ala-Leu-Val-Ala-Val-Ala-Leu-X-X-Ala. Co-translational cleavage of the cell-free product as well as Edman degradation of the stable intracellular form of the protein recovered from Hep G2 cells indicated that this entire 20-amino acid sequence behaved as a signal peptide. There is at least 55% sequence homology between the rat and human apo-A-IV signal peptides and 33% homology between the human A-I and A-IV presegments. Agarose gel chromatography of Hep G2 culture media indicated that neither apo-A-IV nor -A-I is associated with particles that have physical properties resembling any of the plasma lipoprotein density classes. Incubation of plasma with Hep G2 media resulted in transfer of A-I but not A-IV to lipoproteins. Since the NH2 termini of co-translationally cleaved and chylomicron-associated apo-A-IV are identical, it is apparent that 1) this polypeptide does not undergo NH2-terminal post-translational proteolysis like proapo-A-II or proapo-A-I, and 2) regulation of A-IV-lipoprotein interaction is not dependent on any NH2-terminal proteolytic processing event.

Amino Acid Sequence↗

PubMed1984

Rat apolipoprotein A-IV contains 13 tandem repetitions of a 22-amino acid segment with amphipathic helical potential.

Apolipoprotein (apo) A-IV is a 46,000 Mr protein that is a major component of rat high density lipoproteins and chylomicrons. We have isolated, from a rat intestinal cDNA library, a full-length apo A-IV clone of 1423 base pairs and determined its nucleotide sequence. The 1173-nucleotide coding region specifies a protein of 391 amino acids, which includes a 20-amino acid signal peptide. The portion of the cDNA sequence representing the mature plasma protein contains a 66-nucleotide subsequence that is repeated at least 13 times. Although each repeated unit shows some sequence variation, base changes in comparably positioned codons generally conserve the chemical type, if not the identity, of the corresponding amino acids. Nine of the thirteen 22-amino acid repeat units in the derived protein sequence begin with proline and most of these docosapeptides are predicted to have a high content of alpha-helix according to Chou-Fasman rules. When hydration potentials of individual residues are considered, many of the helices are shown to be amphipathic and may thus constitute lipid-binding domains with the ability to activate lecithin:cholesterol acyltransferase. The structure and organization of the repeat units of rat apo A-IV bear a striking similarity to a corresponding repeated sequence block in human apo A-I. This finding raises the possibility of close tandem linkage of the apo A-I and A-IV genes and suggests unequal crossing-over as a mechanism for the evolution of these genes.

Amino Acid Sequence↗