Characterization of the maturation of human pro-apolipoprotein A-I in an in vitro model.

The reaction conditions and the protein structural features involved in the maturation of pro-apolipoprotein A-I (cleavage of pro-peptide) were investigated in an in vitro model. ProapoA-I, mutants and wild type, were expressed in the PGEX/E. coli expression system as fusion proteins with glutathione S-transferase (GST). Use of GST-proapoA-I and truncated forms of proapoA-I enabled quantitation of the amount of GST and apoA-I formed as a result of cleavage following incubation with human serum. Deletion of the pro-peptide (GST-apoA-I) resulted in complete inhibition of the reaction. Truncation of proapoA-I to residues 222, 150, 135, and 25 as well as substitution of residues -6, -5, and -4 with alanine did not affect the reaction. Substitution of residues -1, -2, 1, 3, and 4 with alanine either completely blocked or substantially inhibited cleavage of the pro-peptide. The reaction was inhibited by addition of EDTA, o-phenanthroline, dithiothreitol, and beta-mercaptoethanol and to a lesser extent by p-chloromercuriphenylsulfonic acid, but not by leupeptin, N-ethylmaleimide, PMSF, pepstatin A, or trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane. Calcium was essential for the activation of the cleavage enzyme, but it had a biphasic effect on the cleavage, activating it at concentrations below 1.5 mM and inhibiting at concentrations above 1.75 mM. Manganese alone was not essential for activation of the enzyme nor did it modify the effect of low concentration of calcium. However, a high concentration of manganese partially reverted the inhibitory effect of a high calcium concentration. Thus, residues within -2 to +4 are involved in forming the cleavage site for the maturation enzyme. The reaction of maturation is inhibited by metalloprotease inhibitors and is dependent upon calcium.

Deletion of the propeptide of apolipoprotein A-I reduces protein expression but stimulates effective conversion of prebeta-high density lipoprotein to alpha-high density lipoprotein.

The properties of the mature and pro-forms of recombinant apolipoprotein A-I (apoA-I) were compared with those of apoA-I isolated from human plasma. When the synthesis and secretion of pro- and mature forms of apoA-I from a baculovirus/insect cell expression system were compared in parallel experiments, the amount of the pro-form of apoA-I synthesized and secreted was severalfold higher than that of the mature form of apoA-I. A comparison of the properties of the pro- and mature forms of recombinant apoA-I and human plasma apoA-I showed no difference between all three in their secondary structure, their ability to self-associate, lipid-binding capacity, lecithin: cholesterol acyltransferase activation, and binding to the phospholipid transfer protein. The properties of reconstituted high density lipoprotein (HDL) particles formed from the proteins and their ability to promote cholesterol and phospholipid efflux from human skin fibroblasts were also similar. However, their ability to bind to plasma HDL subfractions differed, because twice as much proapoA-I associated with prebeta(1)-HDL and prebeta(2)-HDL subfractions compared with both mature recombinant and plasma apoA-I. Correspondingly, the amount of proapoA-I in alpha-HDL subfractions, especially in alpha(1)-HDL and alpha(2)-HDL, was decreased. We conclude that while the propeptide of apoA-I is required for the effective synthesis and secretion of apoA-I, cleavage of this peptide is a requisite for the effective interconversion of HDL subfractions.

Binding of phospholipid transfer protein (PLTP) to apolipoproteins A-I and A-II: location of a PLTP binding domain in the amino terminal region of apoA-I.

The interaction of plasma phospholipid transfer protein (PLTP) with HDL has not been characterized in detail, although we have reported that the apoA-I/apoA-II molar ratio in the HDL particle influences PLTP-mediated HDL conversion, but not phospholipid transfer. The aim of this study was to examine whether PLTP binds apoA-I or apoA-II, and if this occurs, then determine the PLTP-binding domain of the apoA-I molecule. To study the PLTP/apolipoprotein interaction we used a solid phase ligand binding assay, the ELISA technique, and apoA-I and apoA-II affinity chromatography. PLTP bound to both apoA-I and apoA-II affinity columns, a finding subsequently utilized in the purification of PLTP. PLTP also bound to both apoA-I and apoA-II on ELISA plates in a concentration-dependent manner, and the binding could be displaced by preincubating the PLTP sample with purified apolipoproteins. To determine which portion of apoA-I is recognized by PLTP, we coated ELISA plates with either recombinant full-length apoA-I or three shortened apoA-I forms sequentially truncated from the C-terminus. To characterize the PLTP binding ability of the C-terminal region of apoA-I, we used both C-terminal CNBr-fragment and a synthetic C-terminal peptide of apoA-I. To further confirm the identity of the binding region, we probed the interaction with a polyclonal and several monoclonal anti-apoA-I antibodies. The antibodies that inhibited the interaction between PLTP and apoA-I were directed towards apoA-I epitopes localized between amino acids 27-141. The polyclonal antibody, R33, and the monoclonal antibody A-I-1 (epitope between amino acids 27-48) were most effective and reduced PLTP binding by 70%. These results show that PLTP binds to both apoA-I and apoA-II, and that the PLTP binding domain of apoA-I resides in the amino terminal region.

Production of mature human apolipoprotein A-I in a baculovirus-insect cell system: propeptide is not essential for intracellular processing but may assist rapid secretion.

To achieve expression of human mature apolipoprotein A-I (apoA-I) in the baculovirus-insect cell expression system, the propeptide encoding region of full-length preproapoA-I was deleted using polymerase chain reaction and the resulting cDNA was cloned into BacPak8 plasmid. After transfection into Sf21 insect cells and plaque purification, mature human apoA-I was secreted by the infected cells into the medium as determined by immunoblotting, amino-terminal sequencing, and molecular weight determination. In both monolayer cell cultures, and in suspension cell culture, maximum expression was achieved by the fifth day. For the first 4 days, 50 to 70% of the synthesized apoA-I was retained in the cells. This intracellular apoA-I was represented by mature apoA-I as shown by immunoblotting and amino-terminal sequencing. Further incubation resulted in a sharp decrease in the cell apoA-I content without a corresponding increase in protein in the medium and most likely represents intracellular degradation of the protein. We conclude that the deletion of the propeptide, while not preventing the correct cleavage of prepeptide during intracellular processing, results in reduced secretion of mature apoA-I. The baculovirus-insect cell expression system described in this study provides a useful method for producing recombinant mature apoA-I and is a potential tool for understanding the function of propeptide in intracellular transport and secretion of apoA-I from cells.

Antibodies against high-density lipoprotein binding proteins enhance high-density lipoprotein uptake but do not affect cholesterol efflux from rat hepatoma cells.

High-density lipoprotein plays a key role in the reverse cholesterol transport pathway as well as in the delivery of cholesterol to the liver and steroidogenic tissues. Metabolism of high-density lipoprotein is determined by one of its apolipoproteins, apolipoprotein A-I; however, the identity and function of cellular protein which binds high-density lipoprotein remains unclear. The effect of antibodies against rat high-density lipoprotein binding proteins, HB1 and HB2, on high-density lipoprotein metabolism in a rat hepatoma cell line were studied. Cells were preincubated with the antibodies and 125I-labeled high-density lipoprotein binding and uptake as well as cholesterol biosynthesis and cholesterol efflux to human plasma or isolated high-density lipoprotein were studied. Both antibodies reacted specifically with HB1 and HB2 on the ligand and Western blots, but their binding was not blocked by high-density lipoprotein. Both antibodies inhibited 125I-labeled high-density lipoprotein binding to cells by 20-40%, but stimulated 125I-labeled high-density lipoprotein uptake by up to 2.5-fold. The antibodies had no effect on cholesterol efflux or on cholesterol synthesis. It is concluded that high-density lipoprotein binding proteins, HB1 and HB2, may be involved in high-density lipoprotein uptake in the liver rather than in mediating cholesterol efflux.

Efflux of cellular cholesterol and phospholipid to apolipoprotein A-I mutants.

Human plasma apolipoprotein A-I (apoA-I) and recombinant full-length proapoA-I (apoA-I-(-6-243)) as well as four truncated forms of proapoA-I were used as acceptors to study cholesterol and phospholipid efflux from HepG2 cells. Efflux of both cholesterol and phospholipid to the lipid-free plasma apoA-I was twice that of apoA-I-(-6-243). When apoA-I was incorporated into reconstituted high density lipoprotein, cholesterol efflux increased, phospholipid efflux decreased and the difference between plasma apoA-I and apoA-I-(-6-243) disappeared. Truncation of recombinant apoA-I to residues 222 (apoA-I-(-6-222)) and 210 (apoA-I-(-6-210)) resulted in a 70-95% decrease in their ability to promote the efflux of both intracellular and plasma membrane cholesterol. Further truncation to residues 150 (apoA-I-(-6-150)) and 135 (apoA-I-(-6-135)) fully restored the ability of apoA-I to promote cholesterol efflux. Phospholipid efflux closely paralleled the efflux of cholesterol. Interaction of 125I-labeled apoA-I with the cells was similar for apoA-I-(-6-243), apoA-I-(-6-222), and apoA-I-(-6-210), but slightly higher for apoA-I-(-6-150) and apoA-I-(-6-135). When complexed with phospholipid, all forms except apoA-I-(-6-210) formed discoidal reconstituted high density lipoprotein particles. When the same amounts of free or lipid-associated apoA-I were compared, association of apoA-I with phospholipid increased cholesterol efflux and decreased phospholipid efflux, and the difference in the ability of different mutants to promote cholesterol and phospholipid efflux disappeared. We conclude that the capacity of lipid-free apoA-I to promote cholesterol efflux is related to its ability to mobilize cellular phospholipid, which apparently involves a region around residues 222-243. A second lipid-binding region is exposed when the carboxyl-terminal half of apoA-I is absent.

Structural and functional properties of full-length and truncated human proapolipoprotein AI expressed in escherichia coli.

Utilizing the Escherichia coli/pGex vector expression system incorporating a thrombin cleavage site, full-length (residues -6-243) and truncated forms of proapolipoprotein AI (proapoAI), terminating at amino acid residues 222, 210, 150, and 135, were purified to levels of at least 5 mg/L, after thrombin cleavage. Assessed by circular dichroism, the helical contents of L-alpha-dimyristoylphosphatidylcholine-associated forms of human plasma-derived apolipoprotein AI (apoAI) and recombinant proapoAI were comparable, being 69% and 65%, respectively. Circular dichroism measurements of the lipid-associated complexes of the truncated forms showed that between the sequence of residues 150-222 no additional helicity was gained until the carboxyl-terminal sequence was present in the molecule, indicating that the carboxyl terminus of the protein is required for the formation of helix within this central region. While tryptophan residues were more than 86% accessible, as assessed by iodide quenching, in the two truncated forms, proapoAI-6-135 and proapoAI-6-150, for both free and complexed protein, this figure fell to about 50% for full-length recombinant proapoAI, further indicating the influence of the carboxyl terminus on the structure of the whole protein. While cross-linking human plasma apoAI in solution with dithiobis-(succinimidyl propionate) revealed high molecular weight oligomers by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, recombinant proapoAI did not strongly form complexes larger than trimers. None of the truncated proapoAI molecules formed oligomers larger than trimers. The shortest form, proapoAI-6-135, only dimerized. Initial results from lecithin:cholesterol acyltransferase activation (apoAI peptide concentration 0.2 microM) indicated that truncation of the 21 carboxy-terminal amino acids resulted in a drop of approximately 53% in activation and 33 residues a drop of 67% relative to the full-length protein. Overall these results indicate the important influence of the carboxyl terminus on the structure of apoAI.

Secretion of biologically active human proapolipoprotein A-I in a baculovirus-insect cell system: protection from degradation by protease inhibitors.

Studies of the structure and function of apolipoprotein A-I (apoA-I) often require its purification by delipidation of high density lipoprotein isolated from large quantities of human plasma and separation of apoA-I from other plasma apolipoproteins. To reduce the need for extensive purification procedures, we have developed an insect cell/baculovirus expression system for the production and secretion of human proapoA-I. The recombinant baculovirus containing full-length human apoA-I cDNA, when introduced into Spodoptera frugiperda, directs the synthesis of preproapoA-I, which is subsequently secreted into the growth medium as proapoA-I, indicating correct processing of the signal peptide during secretion. To prevent the extensive degradation of secreted proapoA-I, leupeptin and pepstatin A were added to the serum free cell culture medium. The protein was simply purified by filtration of the medium, which contained up to 80 mg/l proapoA-I, followed by chromatography on phenyl-sepharose CL-4B. The resultant proapoA-I was found to bind lipid and to activate lecithin:cholesterol acyltransferase as effectively as apoA-I from human plasma. The advantage of this expression system is the ease of purification of intact, biologically active apoA-I in high yield.

Genomic maps of some strains within the Mycoplasma mycoides cluster.

Genomic restriction maps for the small colony (SC) strains (PG1, KH3J, Gladysdale, and V5) of Mycoplasma mycoides subsp. mycoides (the agent of contagious bovine pleuropneumonia) and for Mycoplasma strain PG50 (classified as bovine serogroup 7), with respective sizes of 1,280, 1,280, 1,260, 1,230, and 1,040 kbp, were compared with the map (1,200 kbp) for a large colony strain (Y goat) of M. mycoides subsp. mycoides. The number and order of all mapped restriction sites were fully conserved in the SC genomes, as were the approximate positions of mapped loci. A number of these restriction sites in the Y genome and some, but fewer, in the PG50 genome appeared to be conserved. The SC and large colony strains shared conservation in the relative positions of the mapped loci, except for rpoC.

Human ureaplasmas show diverse genome sizes by pulsed-field electrophoresis.

Contour clamped homogeneous field (CHEF) agarose gel electrophoresis (AGE), ramped to give linear separation of DNA molecules of 600-1600 kilobase pairs (kbp), was used to determine mobilities for full-sized genomic DNA of the serotype standard strains of the human genital mollicutes, Ureaplasma urealyticum relative to yeast chromosomal DNA markers. Indicated genome sizes (in kbp) were 760 for the four biotype 1 strains and 840-1140 for eleven biotype 2 strains. Other estimates were: 720 for Mycoplasma hominis, 1070 for Mycoplasma hyopneumoniae, 890 for Mycoplasma flocculare, 1180 and 1350 for Mycoplasma mycoides subsp. mycoides Y and GC1176-2, respectively, and 1650 and 1580 for Acholeplasma laidlawii B and PG 8, respectively. These data supplement previous evidence from CHEF AGE that the genomes of the Mycoplasmataceae are diverse in size with some larger than previously estimated from DNA renaturation kinetics.

A physical map of the genome of Ureaplasma urealyticum 960T with ribosomal RNA loci.

A physical map is presented for the 900 kilobase pair genome of Ureaplasma urealyticum 960T, locating 29 sites for 6 restriction endonucleases. The large restriction fragments were separated and sized by pulsed-field agarose gel electrophoresis (PFGE). Their locations on the map were determined by probing Southern blots of digests with individual fragments isolated from other digests and by correlating the products of double digestions and partial digestions. An end-labelling technique was used to detect small fragments not readily observed by PFGE. Two loci for rRNA genes have been determined by probing with cloned DNA.

Pulsed-field electrophoresis indicates larger-than-expected sizes for mycoplasma genomes.

The sizes of large DNA fragments produced from genomes of members of the Mycoplasmataceae by digestion with restriction endonucleases having infrequent (1 to 3) cleavage sites within the genome were estimated from their mobility in contour-clamped homogeneous electric field (CHEF) agarose gel electrophoresis by comparison with yeast chromosomal DNA markers. The estimates of total genome size for 7 strains of 6 species ranged from approximately 900 kilo base pairs (kb) for Ureaplasma urealyticum 960T to 1330 kb for M. mycoides subsp. mycoides, GC-1176. The values derived from this new method are considerably higher than those of approximately 500 Mdaltons or 750 kb previously reported for genome sizes in members of the Mycoplasmataceae.

A physical map of the genome of Mycoplasma mycoides subspecies mycoides Y with some functional loci.

A physical map is presented for the 1200 kb genome of Mycoplasma mycoides subsp. mycoides Y, locating 32 cleavage sites for 8 restriction endonucleases. The large restriction fragments involved were separated and sized by pulsed-field agarose gel electrophoresis. Their locations on the map were determined by probing Southern blots of digests with individual fragments isolated from other digests and by correlating the products of double and triple digestions. Loci for 2 ribosomal RNA operons and 2 tRNA operons have been determined by probing with cloned genes and the broad regions of the replication origin and terminus have also been outlined by in vivo labelling studies.

Preparation and FIGE separation of infrequent restriction fragments from Mycoplasma mycoides DNA.

Use of procedures for obtaining satisfactory preparation and digestion of intact DNA of Mycoplasma mycoides subsp. mycoides Y in agarose blocks is reported. The use of inverted field agarose gel electrophoresis (FIGE) for separation of the small number of fragments derived from the genome by several restriction endonuclease digestions is shown. An effect that fragments containing replication forks remain in the well during FIGE, distorting the representative yield of restriction fragments on the gels, is overcome by incubating cells with chloramphenicol for 1 1/2 h before harvest to allow rounds of replication to go to completion without new initiations of DNA synthesis.