Synthesis and secretion of lipoproteins by human hepatocytes in culture

Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [³H]glycerol, more than 92% of the [³H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.     

An approach to the functional analysis of lecithin-cholesterol acyltransferase. Activation by recombinant normal and mutagenized apolipoprotein AI

Apolipoprotein AI (apo AI) of human serum high-density lipoprotein functions as an activator of lecithin-cholesterol acyltransferase (LCAT) and therefore plays an important role in reversed cholesterol transport. The mechanism of the acyltransfer, the activating polypeptide domains of apo AI and the active site of LCAT in this transesterification are not yet known. Synthetic peptides of the apo AI sequence have been designed to determine the activating structure, but did not yet lead to conclusive results. This also applies to spontaneous apo AI mutants. We therefore used the method of site-directed mutagenesis of apo AI cDNAs using the overlap extension approach by the polymerase chain reaction. These constructs were cloned into the procaryotic vector pET8c and expressed under the inducible T7 promoter. The engineered apo AI polypeptides were isolated and purified by affinity chromatography and assayed for their activator activity. The essentials of this approach to the structure and function of activators in general have successfully been exemplified for the LCAT activation by engineering apo AI mutant polypeptides a) by the deletion of two adjacent amphipathic helices (amino acid residues 146-186) and b) by introducing a point mutation (Glu111----Gln).     

Novel human proalbumin variant with intact dibasic sequence facilitates identification of its converting enzyme

We describe here the identification of a new genetic variant of human proalbumin with an N-terminal sequence of Arg-Gly-Val-Phe-Arg-Arg-Val-Ala-His-Lys-. Proalbumin Blenheim (10%) and mature albumin Blenheim (38%) with an initial sequence of Val-Ala-His-Lys-make up nearly half the serum albumin in affected individuals. Despite retaining an intact dibasic processing site, proalbumin Blenheim (1 Asp----Val) enters the circulation unprocessed. The observed ratio of proalbumin to albumin can be accounted for by proteolysis in the periphery. Employed as a potential substrate, proalbumin Blenheim provides a unique means of identifying the physiologically relevant proalbumin convertase. In vitro studies showed that the variant is readily cleaved by trypsin. However, it is not cleaved by the proposed proalbumin convertase, a membrane-bound Ca2+-dependent proteinase prepared from rat liver Golgi vesicles, which gives authentic cleavage of normal human proalbumin.     

Synthesis and processing of human serum apolipoprotein AII in vitro and in Hep G2 cells

The synthesis and structure of the primary translation product of apo AII in a human liver poly(A+) mRNA primed cell-free system and its cotranslational modification was studied parallel to studies in vivo with Hep G2 cells, a human hepatoma cell line. The primary translation product is a preproprotein containing 100 amino acid residues, which is cleaved by the signal peptidase of endoplasmic reticulum to pro-apo AII with the loss of the N-terminal pre-sequence consisting of 18 amino acid residues. Hep G2 cells contain about equal amounts of the proform of apolipoprotein AII and of mature apo AII. Approximately in the same ratio pro- and mature apo AII are secreted into the medium. Determination of the partial amino-acid sequence by automated Edman degradation of the labelled prepro- and proforms of apo AII led to the segmentation of the N-terminus of the primary translation product, consisting of 23 amino acid residues, into the pre-sequence (18 residues) and the pro-sequence (5 residues) with terminal Arg-Arg-residues at the cleavage site to apo AII. We must therefore correct our previously postulated 17 and 6 residues containing segmentation. So far no information has been obtained in which compartment and at what stage of posttranslational events the dimerization occurs by formation of the single disulfide bond at position Cys6 in the mature apo AII structure, leading to the symmetrical molecule.     

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.     

Serum apolipoprotein AI synthesis in rat hepatocytes and its secretion as proform

Rat hepatocytes in monolayer or suspension culture synthesize serum lipoprotein AI. It is secreted into the serum-free culture medium. Synthesis and secretion processes were studied in the presence of radiolabelled amino acids. The synthesis product of the hepatocytes and the secretion product from the medium were isolated by immunoprecipitation with a mono-specific rabbit antiserum against rat apolipoprotein AI. The intracellular and secreted products were homogeneous and identical in polyacrylamide gel electrophoresis but had reduced electrophoretic mobility as compared to native apolipoprotein AI. They were submitted to automated Edman degradation. They were present in their proform, the N-terminus of which is extended by a hexapeptide. In the presence of rat serum the proform is proteolytically transformed into the mature form of apolipoprotein AI.     

Expression of the human serum apolipoprotein AI and AII genes in Xenopus laevis oocytes. Lipid-associated secretion of gene products

The two major apolipoproteins of plasma high-density lipoproteins (HDL) are apolipoprotein AI (apo AI) and AII (apo AII). The apo AI and the correctly oriented apo CIII genes separated by 2.6 kb were obtained by fusion of two human lambda-genomic clones. The apo AII gene was isolated as a 3 kb clone. These apolipoprotein genes have been injected independently and together into Xenopus laevis oocytes and their expression studied. Both apolipoprotein genes were transcribed and translated into their preproforms and processed in Xenopus laevis oocytes to their proforms. They were secreted into the medium associated with newly synthesized phospholipids and neutral lipids as particles floating in the high-density lipoprotein range between 1.12 and 1.21 g/ml. Secreted apo AI is associated mainly with newly synthesized phosphatidylethanolamine and little triglyceride, apo AII with phosphatidylethanolamine, lysophosphatidylethanolamine and neutral lipids. Simultaneous injection of the apo AI and apo AII genes led to the secretion of both apoproteins which separated into two bands during CsCl-density gradient centrifugation. The heavier particles were associated with proapo AI and AII, phosphatidylethanolamine (greater than 90%) and traces of lysophosphatidylethanolamine as lipid components. Proapo AII was immunoprecipitated from the less dense fraction and found to be mainly associated with lysophosphatidylethanolamine. Radiolabelled newly synthesized apolipoproteins in secreted particles were characterized by immunoprecipitation after delipidation of the secreted lipoprotein particles. The oocyte-system proved very suitable for studies of the expression of serum apolipoprotein genes, the assembly of the apolipoproteins with specific lipids to lipoprotein particles and their secretion.     

Conversion of the lipoprotein secreted by cultured eel hepatocytes to high density lipoprotein

• 1.1. The lipoprotein, a VLDL-like lipoprotein, secreted by cultured eel hepatocytes was incubated with whole eel serum, serum HDL, or serum VLDL. No change in the VLDL-like lipoprotein was found.
• 2.2. The secreted lipoprotein was incubated with five kinds of liposomes and a HDL-like particle was formed in the presence of BSA only when l-α-dimyristoyl lecithin liposome was used.
• 3.3. In the presence of 3% BSA, apo AI, proapo AI, apo AII and apo C of the secreted lipoprotein were transferred to the l-α-dimyristoyl lecithin liposome and a HDL-like particle was formed.
• 4.4. The secreted lipoprotein was hydrolyzed by lipoprotein lipase and a HDL-like particle formed after hydrolysis contained no triglyceride and had phospholipid as its main lipid.

     

Acute suppression of apo B secretion by insulin occurs independently of MTP

Secretion of apolipoprotein (apo) B-containing lipoproteins by the liver depends mainly upon apo B availability and microsomal triglyceride transfer protein (MTP) activity and is subject to insulin regulation. Hepatic MTP mRNA expression is negatively regulated by insulin which correlates with inhibition of apo B secretion suggesting that insulin might suppress apo B secretion through an MTP-dependent mechanism. To investigate this possibility, we examined the acute effect of insulin on hepatic MTP expression and activity levels in vivo utilizing apobec-1^−/− mice. Insulin did not significantly alter hepatic MTP mRNA levels or lipid transfer activity 2 h following injection, but suppressed expression of genes important in gluconeogenesis. To study the specific role of MTP, we expressed human MTP (hMTP) in primary rat hepatocytes using adenoviral gene transfer. Increased expression of hMTP resulted in a 47.6 ± 17.9% increase in total apo B secreted. Incubation of hepatocytes with insulin suppressed apo B secretion by 50.1 ± 10.8% in cells over-expressing hMTP and by 53.0 ± 12.4% in control transfected hepatocytes. Results indicate that even under conditions of increased hepatic apo B secretion mediated by MTP, responsiveness of hepatocytes to insulin to suppress apo B secretion is maintained.     

Distinct importin recognition properties of histones and chromatin assembly factors

The adhesive protein vitronectin (75 kDa) occurs in human blood fluid in a one-chain (Vn₇₅) or a two-chain form (Vn₆₅₊₁₀), and is produced by a specific cleavage (at Arg³⁷⁹–Ala³⁸⁰), by a proteinase not identified hitherto. These two forms were shown to be functionally different and therefore, this cleavage may have a regulatory significance in vivo. Here, we report the use of a tailored one-chain recombinant Vn, a specific protein kinase A phosphorylation at Ser³⁷⁸, and sequence analysis to show: (1) that none of the proteinases originating from blood, previously thought to be the endogenous proteinase (plasmin, thrombin, tPA, and uPA), is indeed the in vivo convertase; and (2) that furin, a serine endoproteinase residing in the secretory pathway of hepatocytes, where Vn is synthesized, specifically cleaves Vn at the endogenous cleavage site. Consequently, we propose that the Vn₇₅ to Vn₆₅₊₁₀ conversion takes place in the liver (not in blood) and is carried out by furin.     

Differences in local conformation in human apolipoprotein B-100 of plasma low density and very low density lipoproteins as identified by cathepsin D

The conformational changes of human apolipoprotein (apo) B-100 which accompany the conversion of plasma very low density lipoproteins (VLDL) to low density lipoproteins (LDL) were investigated by studying the accessibility of apoB-100 in LDL and VLDL to limited proteolysis with cathepsin D, an aspartyl proteinase involved in intracellular protein degradation. We characterized the proteolytic products of apoB-100 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by NH2-terminal sequence analysis to locate cleavage sites. The results identified at least 10 cleavage products generated from apoB-100 and showed differential accessibility of cleavage sites for cathepsin D in apoB-100 between LDL and VLDL. We identified a specific peptide region (residues 2660-2710), which is preferentially accessible to limited proteolysis by cathepsin D but inaccessible to limited proteolysis by 12 other enzymes tested. Within this peptide region, cathepsin D cleaved apoB-100 of LDL and VLDL preferentially at different sites, separated by 33-36 amino acids (2665-2666 or 2668-2669 (LDL) and 2701-2702 (VLDL]. In addition, we identified a cleavage site, located at residues 3272-3273, specific for cathepsin D, which is contained within the COOH-terminal enzyme-accessible peptide region (residues 3180-3280), which we have demonstrated using 12 endoproteases with various specificities. The previously identified NH2-terminal region (residues 1280-1320) appears to be resistant to limited cleavage by cathepsin D. However, a new site was revealed only approximately 66 kDA from the NH2 terminus. We conclude that differential accessibility and the shift of the novel scission site for cathepsin D by 33-36 amino acids indicate significant differences in local conformation at these sites in apoB-100 as VLDL are converted to LDL.     

Synthesis, transport, and processing of apolipoproteins of high density lipoproteins

Cell biology methods have greatly influenced the elucidation of the biosynthetic pathways of apolipoproteins. In vitro and tissue culture systems allow the study, to a large extent, of the process of synthesis, intracellular processing, secretion, and extracellular processing of the major high density lipoprotein apoproteins apoA-I and A-II and also of a minor component, apoA-IV. Whereas the latter apoprotein is equipped only with a signal sequence, the primary translation products of apoA-I and apoA-II carry N-terminal extensions of preprosequence of 24 amino acids for apoA-I and 23 amino acid residues for apoA-II. The pro-form of apoA-I characterized by a hexapeptide extension is completely stable intracellularly and is secreted as such. The pro-form is further processed by a serum protease specific for an unusual -Gln-Gln-Asp-Glu-sequence site. Pro-apoA-II, a pentapeptide sequence, is partially processed intracellularly to its mature form and secreted together with the residual pro-form. The cleavage site of pro-apoA-II is characterized by two basic amino acid residues Arg-Arg, present also in other known pro-proteins. The biological function of the N-terminal pro-sequences and details of their final processing by the serum protease(s) have yet to be established.     

利用大肠杆菌高效表达人载脂蛋白AI及其性质分析

载脂蛋白AI(apolipoproteinAI,apoAI)是高密度脂蛋白HDL的主要组成成分 ,流行病学研究表明apoAI的含量决定血浆中HDL的高低 ,而HDL具有胆固醇逆向转运的功能 ,起到降低冠心病发生概率的作用。通过大肠杆菌表达系统来生产apoAI的蛋白原proapoAI,蛋白的表达形式为包涵体 ,通过疏水柱进行柱上复性。同时在proapoAI和apoAI之间设计一个酸水解位点 ,通过将蛋白原proapoAI进行酸水解来得到成熟蛋白apoAI。最终得到的成熟蛋白apoAI在结构分析和脂结合特性上都与天然的apoAI相似 ,从而为该蛋白的工业生产上提供了一个良好的基础。     

Transient expression of wild type and mutant human apolipoprotein AI in COS cells

A human apolipoprotein AI (apo AI) minigene and two mutants were cloned into the vector pUHD10-1 for expression studies in COS cells under the control of the strong CMV (cytomegalovirus) enhancer and the own apo AI promoter. In the mutated apo AI minigene (mutant M1) the positions of the triplets of Gln(-2)-Gln-1 at the C-terminus of the prosequence were exchanged against Gln(-8)-Ala-7, the recognition site of the signal peptidase of the wild type human apo AI. The prosequence has been deleted in mutant M2 and the presequence linked directly to the N-terminus of the mature apo AI form. We report here on expression studies in COS cells, a cell line, which does not express apo AI. They were transfected by electroporation with pUHD10-1 constructs, which contain a) the wild type apo AI minigene and b) the two mutant apo AI minigenes with mutations described above. The following results were obtained: a) the wild type and mutant apo AI constructs were efficiently transcribed and translated in COS cells, b) the expression of the wild type preproapo AI minigene in COS cells led to the secretion of proapo AI (29 kDa), that of the mutant (M2) gene, devoid of the prosequence of mature apo AI (28.4 kDa), whereas the product of mutant gene M1 (31 kDa) with the recognition site of the signal peptides transposed to the C-terminus of the prosequence remained uncleaved within the COS cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping of the Feline Calicivirus Proteinase Responsible for Autocatalytic Processing of the Nonstructural Polyprotein and Identification of a Stable Proteinase-Polymerase Precursor Protein

Expression of the region of the feline calicivirus (FCV) ORF1 encoded by nucleotides 3233 to 4054 in an in vitro rabbit reticulocyte system resulted in synthesis of an active proteinase that specifically processes the viral nonstructural polyprotein. Site-directed mutagenesis of the cysteine (Cys1193) residue in the putative active site of the proteinase abolished autocatalytic cleavage as well as cleavage of the viral capsid precursor, suggesting that this "3C-like" proteinase plays an important role in proteolytic processing during viral replication. Expression of the region encoding the C-terminal portion of the FCV ORF1 (amino acids 942 to 1761) in bacteria allowed direct N-terminal sequence analysis of the virus-specific polypeptides produced in this system. The results of these analyses indicate that the proteinase cleaves at amino acid residues E960-A961, E1071-S1072, E1345-T1346, and E1419-G1420; however, the cleavage efficiency is varied. The E1071-S1072 cleavage site defined the N terminus of a 692-amino-acid protein that contains sequences with similarity to the picornavirus 3C proteinase and 3D polymerase domains. Immunoprecipitation of radiolabeled proteins from FCV-infected feline kidney cells with serum raised against the FCV ORF1 C-terminal region showed that this "3CD-like" proteinase-polymerase precursor protein is apparently stable and accumulates in cells during infection.     

Cleavage of cartilage proteoglycan between G1 and G2 domains by stromelysins

Normal and pathological turnover of proteoglycans in articular cartilage involves its cleavage close to the N-terminal G1 domain responsible for aggregation. A fragment containing G1 and G2 N-terminal domains of pig cartilage proteoglycans was therefore used as a substrate to investigate its degradation by the metalloproteinase stromelysin and related recombinant stromelysin enzymes. The stromelysins produced an apparent single cleavage yielding a G1 fragment of 56 kDa and a G2 fragment of 110 kDa. Rabbit bone stromelysin was much more active against the G1-G2 fragment and against proteoglycan aggregates than recombinant human stromelysin-1 and stromelysin-2. All metalloproteinase preparations were active against proteoglycan and the G1-G2 fragment at acid (pH 5.5) and neutral pH (7.4). N-terminal sequencing of the G2 fragment derived from the action of recombinant human stromelysin-1 revealed that cleavage between G1 and G2 occurred at the N-terminal end of the interglobular domain, close to the last cysteine in G1. The specific cleavage site was between an asparagine and a pair of phenylalanine residues, where the asparagine corresponds to residue 341 in human and rat mature core protein sequence.