Sedimentation velocity analysis of amyloid oligomers and fibrils.

The different aggregation states of amyloid oligomers and fibrils have been associated with distinct biological properties and disease pathologies. These various amyloid species are distinguished by their different molecular weights and sedimentation coefficients and can be consistently resolved, separated, and analyzed using sedimentation velocity techniques. We first describe the theoretical background and use of the preparative ultracentrifuge to separate amyloid fibrils and their oligomeric intermediates from monomeric subunits as well as the factors and limits involved in such methods. The approach can be used to monitor the kinetics of fibril formation as well as providing purified fractions for functional analysis. Secondly, we describe the use of analytical ultracentrifugation as a precise and robust system for monitoring the rate of sedimentation of amyloid fibrils under different solution conditions. Sedimentation velocity procedures to characterize the size, interactions, and tangling of amyloid fibrils as well as the binding of nonfibrillar components to form heterologous complexes are detailed.

Non-fibrillar components of amyloid deposits mediate the self-association and tangling of amyloid fibrils.

Amyloid deposits are proteinaceous extra-cellular aggregates associated with a diverse range of disease states. These deposits are composed predominantly of amyloid fibrils, the unbranched, beta-sheet rich structures that result from the misfolding and subsequent aggregation of many proteins. In addition, amyloid deposits contain a number of non-fibrillar components that interact with amyloid fibrils and are incorporated into the deposits in their native folded state. The influence of a number of the non-fibrillar components in amyloid-related diseases is well established; however, the mechanisms underlying these effects are poorly understood. Here we describe the effect of two of the most important non-fibrillar components, serum amyloid P component and apolipoprotein E, upon the solution behavior of amyloid fibrils in an in vitro model system. Using analytical ultracentrifugation, electron microscopy, and rheological measurements, we demonstrate that these non-fibrillar components cause soluble fibrils to condense into localized fibrillar aggregates with a greatly enhanced local density of fibril entanglements. These results suggest a possible mechanism for the observed role of non-fibrillar components as mediators of amyloid deposition and deposit stability.

Human apoC-IV: isolation, characterization, and immunochemical quantification in plasma and plasma lipoproteins.

Apolipoprotein C-IV (apoC-IV), the newest member of the low-molecular-weight apoC group, has been characterized in blood plasma of rabbits, in which it is a major proline-rich apoC component (Zhang, L-H., L. Kotite, and R. J. Havel. 1996. Identification, characterization, cloning, and expression of apoC-IV, a novel sialoglycoprotein of rabbit plasma lipoproteins. J. Biol. Chem. 271: 1776-1783). Although the decoded sequence of mouse and human apoC-IV is known, apoC-IV has not been identified in blood plasma from these or other species. Rabbit apoC-IV exists in several sialoforms, and the asialoform has an acidic isoelectric point. We show that apoC-IV is a basic protein in human, monkey, and mouse plasma, present as a minor apoC component of VLDL. Human apoC-IV, isolated from apo VLDL by DEAE-cellulose chromatography and two-dimensional electrophoresis, was identified by microsequencing four tryptic peptides. The protein exhibits two major isoforms; one is N-glycosylated, and both are variably sialylated. In normolipidemic plasma, greater than 80% of the protein is in VLDL (0.7% of total apo VLDL), with most of the remainder in HDL. The concentration of apoC-IV in the plasma and lipoproteins of rho < 1.21 g/ml is closely related to plasma triglyceride concentration up to 1,770 mg/dl, varying from 0.1-1.9 mg/dl. Neither the human nor rabbit apoC-IV gene contains a typical TATA box in the 5'-flanking region, but the 5'-untranslated region of the rabbit gene contains a unique purine-rich sequence, GGGACAG(G/A), repeated nine times in tandem, with an additional two within the 5'-flanking sequence. This sequence, functioning as a GAGA box that has been implicated in the transcription of a number of genes, may explain the higher level of expression of apoC-IV in rabbits.

Cross-linking and amyloid formation by N- and C-terminal cysteine derivatives of human apolipoprotein C-II.

We have investigated the effect of disulfide cross-linking on amyloid formation by human apolipoprotein (apo) C-II. Three derivatives of apoC-II were generated by inserting a cysteine residue on either the N-terminus (C(N)-apoC-II), C-terminus (C(C)-apoC-II), or both termini (C(N)C(C)-apoC-II). Under reducing conditions, all derivatives formed amyloid with a fibrous ribbon morphology similar to that of wild-type apoC-II. Under oxidizing conditions, C(N)- and C(N)C(C)-apoC-II formed a highly tangled network of fibrils, suggesting that the addition of an N-terminal cysteine to apoC-II promotes interfibril disulfide cross-links. Fibrils formed by C(C)-apoC-II under oxidizing conditions were closely packed but less tangled than fibrils formed by the C(N) and C(N)C(C) derivatives. The frequency of closed ring structures was more than doubled for C(C)-apoC-II compared to wild-type apoC-II. The kinetics of fibril formation by all cysteine derivatives was markedly enhanced under oxidizing conditions, suggesting that disulfide cross-linking promotes amyloid formation. Substoichiometric levels of preformed C(N)- and C(C)-apoC-II dimers accelerate amyloid formation by wild-type apoC-II. These data suggest that the N- and C-termini of apoC-II are close together in the amyloid fibril such that covalent cross-linking of either the N or C end of apoC-II promotes nucleation and the "seeding" of fibril growth.

Functional analyses of human apolipoprotein CII by site-directed mutagenesis: identification of residues important for activation of lipoprotein lipase.

Apolipoprotein CII (apoCII) activates lipoprotein lipase (LPL). Seven residues, located on one face of a model alpha-helix spanning residues 59-75, are fully conserved in apoCII from ten different animal species. We have mutated these residues one by one. Substitution of Ala(59) by glycine, or Thr(62) and Gly(65) by alanine did not change the activation, indicating that these residues are outside the LPL-binding site. Replacement of Tyr(63), Ile(66), Asp(69), or Gln(70) by alanine lowered the affinity for LPL and the catalytic activity of the LPL-apoCII complex. For each residue several additional replacements were made. Most mutants retained some activating ability, but replacement of Tyr(63) by phenylalanine or tryptophan and Gln(70) by glutamate caused almost complete loss of activity. All mutants bound to liposomes with similar affinity as wild-type apoCII, and they also bound with similar affinity to LPL in the absence of hydrolyzable lipids. However, the inactive mutants did not compete with wild-type apoCII in the activation assay. Therefore, we conclude that the productive apoCII-LPL interaction may be dependent on substrate molecules. In summary, our data demonstrate that residues 63, 66, 69, and 70 are of special importance for the function of apoCII, but no single amino acid residue is absolutely crucial.

The tree shrew apolipoprotein C-I cDNA: sequence and its expression.

A rabbit anti-serum to tree shrew apolipoprotein C-I (apo C-I) was used to screen an expression cDNA library constructed by us from tree shrew (TS) liver tissue. Two apo C-I cDNA clones were obtained. The longer one consists of 380 nucleotides, including 21 bp and 95 bp at the 5' and 3' end of the non-translated regions respectively, and a 264-bp fragment in an open reading frame encoding 88 amino acids prepropeptide which contains 26 amino acids of signal peptide and a mature protein (62 amino acids). Comparing the amino-acid sequence deduced from this cDNA with those of the published mammalian apo C-Is reveals that it shared some structural similarity with rat, mouse and dog apo C-I, but it had 5 more amino acids than that of human and baboon. The expression of apo C-I mRNA in 8 different tissues were also assayed with Northern blot. The results demonstrated that liver had the highest expression, intestine had much less expression and no expression in other tissues, which is much different from human and other species. This study has laid down a good foundation for further studying on the function and the stucture of tree shrew apo C-I gene.

Apolipoprotein CII from rainbow trout (Oncorhynchus mykiss) is functionally active but structurally very different from mammalian apolipoprotein CII.

Apolipoprotein CII (apoCII) plays an important role in plasma lipid metabolism as an activator for lipoprotein lipase (LPL). We have amplified and sequenced apoCII cDNA from rainbow trout. Amino acid sequence analyses confirmed that this sequence corresponded to the protein that had apoCII activity. Northern blot analyses showed that apoCII mRNA was present in both liver and intestine, but the level in intestine was very low. Two major transcripts (800 and 600bp) were found. The predicted amino acid sequence consists of 112 amino acid residues, including the signal peptide. The mature peptide is seven residues longer than human apoCII (86 versus 79 residues) due to an extension at the amino-terminal end. The rainbow trout sequence showed an overall identity of only 20-25% to previously known apoCII sequences. The carboxy-terminal region (residues 51-79, human numbering) showed 35-45% identity to other apoCII sequences, while in the amino-terminal region, there was little if any identity and it was not possible to predict any long amphipathic, potentially lipid-binding alpha-helices. Trout apoCII was present in all lipoprotein fractions including LDL. At +10 degrees C trout plasma showed higher ability to stimulate LPL than human plasma. We conclude that apoCII from rainbow trout is in most parts structurally different from apoCII from other species, and that it is adapted to function at low temperature.

Characterization of recombinant wild type and site-directed mutations of apolipoprotein C-III: lipid binding, displacement of ApoE, and inhibition of lipoprotein lipase.

The physicochemical properties of recombinant wild type and three site-directed mutants of apolipoprotein C-III (apoC-III), designed by molecular modeling to alter specific amino acid residues implicated in lipid binding (L9T/T20L, F64A/W65A) or LPL inhibition (K21A), were compared. Relative lipid binding efficiencies to dimyristoylphosphatidylcholine (DMPC) were L9T/T20L > WT >K21A > F64A/W65A with an inverse correlation with size of the discoidal complexes formed. Physicochemical analysis (Trp fluorescence, circular dichroism, and GdnHCl denaturation) suggests that L9T/T20L forms tighter and more stable lipid complexes with phospholipids, while F64A/W65A associates less tightly. Lipid displacement properties were tested by gel-filtrating apoE:dipalmitoylphosphatidylcholine (DPPC) discoidal complexes mixed with the various apoC-III variants. All apoC-III proteins bound to the apoE:DPPC complexes; the amount of apoE displaced from the complex was dependent on the apoC-III lipid binding affinity. All apoC-III proteins inhibited LPL in the presence or absence of apoC-II, with F64A/W65A displaying the most inhibition, suggesting that apoC-III inhibition of LPL is independent of lipid binding and therefore of apoC-II displacement. Taken together. these data suggest that the hydrophobic residues F64 and W65 are crucial for the lipid binding properties of apoC-III and that redistribution of the N-terminal helix of apoC-III (L9T/T20L) enhances the stability of the lipid-bound protein, while LPL inhibition by apoC-III is likely to be due to protein:protein interactions.

Apolipoprotein C-III displacement of apolipoprotein E from VLDL: effect of particle size.

ApoC-III and apoE are important determinants of intravascular lipolysis and clearance of triglyceride-rich chylomicrons and VLDL from the blood plasma. Interactions of these two apolipoproteins were studied by adding purified human apoC-III to human plasma at levels observed in hypertriglyceridemic subjects and incubating under specific conditions (2 h, 37 degrees C). As plasma concentrations of apoC-III protein were increased, the contents in both VLDL and HDL were also increased. Addition of apoC-III at concentrations up to four times the intrinsic concentration resulted in the decreasing incremental binding of apoC-III to VLDL while HDL bound increasing amounts without evidence of saturation. No changes were found in lipid content or in particle size of any lipoprotein in these experiments. However, distribution of the intrinsic apoE in different lipoprotein particles changed markedly with displacement of apoE from VLDL to HDL. The fraction of VLDL apoE that was displaced from VLDL to HDL at these high apoC-III concentrations varied among individuals from 20% to 100% its intrinsic level. The proportion of VLDL apoE that was tightly bound (0% to 80%) was found to be reproducible and to correlate with several indices of VLDL particle size. In the group of subjects studied, strongly adherent apoE was essentially absent from VLDL particles having an average content of less than 50,000 molecules of triglyceride. Addition of apoC-III to plasma almost completely displaces apoE from small VLDL particles. Larger VLDL contain tightly bound apoE which are not displaced by increasing concentration of apoC-III.

Semi-automated rapid isoelectric focusing of apolipoproteins C from human plasma using Phastsystem and immunofixation.

Apolipoproteins (apo) C-I, C-II, and C-III play crucial roles in intravascular lipid metabolism. Whereas apo C-II is an obligate cofactor for lipoprotein lipase, apo C-III was shown to inhibit its action. Apo C-I can be a potent cofactor of human lecithin:cholesterol acyltransferase. Structural mutants and deficiencies of apo C-II lead to hypertriglyceridemia. A similar phenotype is associated with apo C-III mutants and is inducible by overexpression of human apo C-III in transgenic animals. No structural variant has so far been reported for apo C-I. The present paper describes a rapid semi-automated procedure for isoelectric focusing analysis of these C-apolipoproteins from whole plasma or serum and their visualization by immunofixation and silver staining. The procedure allows detection of charged variants of C-apolipoproteins. As applied to 295 patients with coronary heart disease and 85 controls, it also serves to detect deficiency syndromes of these apolipoproteins. The procedure provides reliable, easy and quick analysis of C-apolipoproteins applicable as a routine or screening procedure not restricted to specialized laboratories.

[Isolation of isoforms of apolipoprotein CIII from human serum by chromatofocusing].

This study aimed to isolate isoforms of apolipoprotein (apo) C III from human serum. 24-hour fasting serum from normal and hyperlipidemic subjects was pooled and subjected to ultracentrifugation at plasma density for 20 hours. Very low density lipoprotein (VLDL) was collected at density of d < 1.006 g/ml, and it was delipidated by ethanol and ether. The delipidated apo-VLDL was dissolved in a solution containing 7.2 mol/L urea and 20 mmol/L dithiothreitol. The insoluble apo B was removed by centrifugation. The soluble apo-VLDL was applied to PBE94 column, and eluted with elution buffer containing polybuffer 74 and 8 mol/L urea (1:8, pH4.0). After pooled, the eluted peaks of apolipoproteins were applied to column chromatography of hydroxylapatite to remove the polybuffer. The purified isoforms of apoC III and the purified apo C I, C II and E, were characterized by isoelectrofocusing and west blot. The results showed that the purified apoC III1, C III2, and C II were pure.

Identification and purification of apolipoprotein C-III from the serum of cows.

OBJECTIVE: To identify, purify, and analyze distribution of apolipoprotein C-III in lipoprotein fractions and to evaluate its concentration in serum from calves, heifers, and cows during various stages of lactation. SAMPLE POPULATION: Sera from 3 female calves, 3 heifers, and 12 cows during early, middle, late, and nonlactation stages. PROCEDURE: Apolipoprotein C-III was identified by use of amino-terminal amino acid sequence analysis of bands separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purification was performed by extraction with acetone, delipidation with 2-propanol, and 2 chromatographic steps. The apolipoprotein C-III concentration in the total lipoprotein fraction was evaluated by densitometric scanning of the bands for apolipoprotein C-III separated by electrophoresis, using purified apolipoprotein C-III as an internal standard. RESULTS: Apolipoprotein C-III was identified as 8.2- and 7.3-kd proteins with identical amino acid sequences. The 2 proteins were mainly found in the high-density lipoprotein fraction, then were purified separately. Serum apolipoprotein C-III concentration was significantly (P < 0.05) higher in cows during lactation than in nonlactating cows and was negatively correlated with serum triglyceride concentration (r = -0.620, P < 0.01). CONCLUSIONS: The role of apolipoprotein C-III in cows may involve a function related to lactation.

Isolation and characterization of recombinant human apolipoprotein C-II expressed in Escherichia coli.

A full-length recombinant human apolipoprotein C-II (ApoC-II) has been successfully expressed in Escherichia coli using the T7 expression system. The recombinant ApoC-II. which was expressed intracellularly in the inclusion bodies, was solubilized with 8 M urea and purified using Sephadex G-75 gel permeation chromatography. Four liters of the bacterial culture yielded 16-20 mg of purified recombinant ApoC-II. Sequencing and mass spectrometric analyses indicated that the isolated recombinant ApoC-II contained predominantly (64%) the native form with threonine as the N-terminus, but also contained a minor (36%) molecular form of ApoC-II with an additional methionine at the N-terminus (Met-ApoC-II). Analysis of the recombinant ApoC-II by tryptic digestion and high performance liquid chromatography-electrospray mass spectrometry provides additional conclusive evidence that, with the exception of the N-terminus of Met-ApoC-II, the expressed ApoC-II has the expected peptide sequence. However, this extra N-terminal methionine residue can be excised by further in vitro treatment with methionine aminopeptidase. The purified recombinant ApoC-II was found to be competent in the activation of bovine milk lipoprotein lipase. Thus, the recombinant ApoC-II prepared from E. coli may have a pharmacological application for the treatment of patients with genetic hypertriglyceridemia caused by ApoC-II deficiency.

Evaluation of double filtration plasmapheresis, thermofiltration, and low-density lipoprotein adsorptive methods by crossover test in the treatment of familial hypercholesterolemia patients.

A comparative assessment has been made regarding efficacy and safety of the double filtration plasmapheresis (DFPP), thermofiltration (TFPP), and low-density lipoprotein (LDL) adsorptive (PA) methods by making a crossover test on heterozygous familial hypercholesterolemia patients. Treatments by DFPP, TFPP (secondary membrane Evalux 5A), and PA (Liposorber LA-40) were carried out 5 times each, with a 2-week interval, in 5 patients with heterozygous familial hypercholesterolemia. The same plasma separator (Plasmacure PS-60, polysulfone) was used in all cases, and the volume of plasma processed was set at 4 L. High removal rates were obtained of total cholesterol, LDL cholesterol, triglycerides TG, and apolipoprotein B (apoB) by all three methods, and no differences were observed. Lipoprotein (a), apoA-2, apoC-3, fibrinogen, and immunoglobulin M (IgM) showed significantly high removal rates by the DFPP and TFPP methods compared with the PA method. The sieving coefficient of albumin and high-density lipoprotein (HDL) cholesterol at 2 and 4 L of plasma processed exhibited high permeabilities using all three methods. Supplementing albumin was not necessary. An increase of the transmembrane pressure was observed in 1 case treated by DFPP but was not observed when using the TFPP or PA method. No changes were observed in serum interleukin 1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha) before and after treatment by any of the three methods. No remarkable side effects were observed using either the DFPP or TFPP method. The DFPP and TFPP methods showed efficacy and safety that was not inferior to the PA method in conventional LDL apheresis, and the dead-end method of the filter operation without the discarding of plasma was shown to be possible.

Identification, characterization, cloning, and expression of apolipoprotein C-IV, a novel sialoglycoprotein of rabbit plasma lipoproteins.

We have identified and characterized a novel proline- and arginine-rich protein component of lipoproteins, present in up to five sialylated isoforms, in rabbit blood plasma. The pI of the desialylated protein is 5.7. Based upon its N-terminal sequence, a complete cDNA sequence of 555 nucleotides was cloned from rabbit liver. The synthesized protein is predicted to contain 124 amino acids, including a typical signal peptide of 27 residues. The mature protein of 97 amino acids, designated apolipoprotein C-IV, is associated with the lipoproteins of blood plasma, primarily very low density and high density lipoproteins. It contains two potential amphipathic helices characteristic of plasma apolipoproteins and forms discoidal micelles with phosphatidylcholine. Northern analysis shows a single 0.6-kilobase apolipoprotein C-IV mRNA, detected only in the liver, and Southern analysis suggests a single copy gene. Sialylated apolipoprotein C-IV is secreted from transfected mammalian cells. Nucleotide sequence comparisons demonstrate a strong homology to portions of the upstream regions of the mouse and human apolipoprotein C2 genes, within each of which a distinct gene has recently been identified. The nucleotide sequences and the predicted amino acid sequences, as well as corresponding cDNA sequences in the rat and monkey, indicate that the apolipoprotein C4 gene has been highly conserved during mammalian evolution.

Crystallization and preliminary X-ray analysis of human plasma apolipoprotein C-I.

Human apolipoprotein C-I, the smallest of the plasma apolipoproteins, is a component of several classes of plasma lipoproteins. Two crystalline forms of this protein, both suitable for high resolution X-ray diffraction analysis, have been obtained from mixtures of 2-methyl-2,4-pentanediol, sodium acetate, and octyl-beta-D-1-thioglucopyranoside at room temperature. The first form belongs to space group P2(1), with beta = 106 degrees and cell dimensions of a = 38.1 A, b = 50.8 A and c = 34.9 A. The asymmetric unit appears to contain two molecules of the protein. The second form belongs to space group P2(1)2(1)2(1), with cell dimensions of a = 34.5 A, b = 53.1 A, c = 71.1 A and it also appears to contain two molecules of the protein in the asymmetric unit.

[Separation and purification of human apolipoproteins A-I and C-III by chromatofocusing].

Human very low density lipoprotein (VLDL) and high density lipoprotein (HDL) were isolated and purified by a process of combined dextran sulfate precipitation and density gradient ultracentrifugation. Chromatofocusing, which separates protein based on differences in isoelectric point, was used to separate apolipoprotein A-I (apoA-I) and apolipoprotein C-III from human HDL and VLDL, respectively. Discontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analytical isoelectric focusing (IEF) were used to study the purity of different fractions. Both purified apoA-I and apoC-III showed single bands on SDS-PAGE at molecular weights of 28183 and 9400 Daltons, respectively. As determined by IEF in the presence of 8 mol/L urea, apoA-I had eight isoforms with pI of 5.66-5.87. The pI's of the three isoproteins of apoC-III (C-III0, C-III1 and C-III2) were 5.06, 4.88 and 4.72, respectively. Chromatofocusing, a new simple technique combining the high resolving power of IEF with the high capacity of ion-exchange column chromatography, is extremely valuable for large-scale purification of the major apolipoproteins of VLDL and HDL.

A missense mutation (Trp 26-->Arg) in exon 3 of the apolipoprotein CII gene in a patient with apolipoprotein CII deficiency (apo CII-Wakayama).

We studied the molecular basis of a case of apolipoprotein CII (apo CII) deficiency with a history of familial consanguinity. DNA sequence analysis of the apo CII gene from the patient revealed a homozygous nucleotide change: a T-->C transition for codon 26 (TGG) at nucleotide 2967 of the third exon resulting in a Trp26-->Arg substitution. His mother was heterozygous of the same mutation and showed half the value of normal apo CII/apo CIII. Analysis of his brother who showed the normal apo CII concentration revealed no mutation at the same place. These results suggested that this missense mutation could be the cause of apo CII deficiency in this kindred.