Lawrence Chan

Structure and evolution of the apolipoprotein multigene family

We present the complementary DNA and deduced amino acid sequence of rat apolipoprotein A-II (apoA-II), and the results of a detailed statistical analysis of the nucleotide and amino acid sequences of all the apolipoprotein gene sequences published to date: namely, those of human and rat apoA-I, apoA-II and apoE, rat apoA-IV, and human apoC-I, C-II and C-III. Our results indicate that the apolipoprotein genes have very similar genomic structures, each having a total of three introns at the same locations. Using the exon/intron junctions as reference points, we have obtained an alignment of the coding regions of all the genes studied. It appears that the mature peptide regions of these genes are almost completely made up of tandem repeats of 11 codons. The part of mature peptide region encoded by exon 3 contains a common block of 33 codons, whereas the part encoded by exon 4 contains a much more variable number of internal repeats of 11 codons. These genes have apparently evolved from a primordial gene through multiple partial (internal) and complete gene duplications. On the basis of the degree of homology of the various sequences, and the pattern of the internal repeats in these genes, we propose an evolutionary tree for the apolipoprotein genes and give rough estimates of the divergence times between these genes. Our results show that apoA-II has evolved extremely rapidly and that apoA-I and apoE also have evolved at high rates but some regions are better conserved than the others. The rate of evolution of individual regions seems to be related to the stringency of their functional requirements.

Hyperhomocysteinemia Decreases Circulating High-Density Lipoprotein by Inhibiting Apolipoprotein A-I Protein Synthesis and Enhancing HDL Cholesterol Clearance

We previously reported that hyperhomocysteinemia (HHcy), an independent risk factor of coronary artery disease (CAD), is associated with increased atherosclerosis and decreased plasma high-density lipoprotein cholesterol (HDL-C) in cystathionine &bgr;-synthase–/apolipoprotein E–deficient (CBS−/−/apoE−/−) mice. We observed that plasma homocysteine (Hcy) concentrations are negatively correlated with HDL-C and apolipoprotein A1 (apoA-I) in patients with CAD. We found the loss of large HDL particles, increased HDL-free cholesterol, and decreased HDL protein in CBS−/−/apoE−/− mice, and attenuated cholesterol efflux from cholesterol-loaded macrophages to plasma in CBS−/−/apoE−/− mice. ApoA-I protein was reduced in the plasma and liver, but hepatic apoA-I mRNA was unchanged in CBS−/−/apoE−/− mice. Moreover, Hcy (0.5 to 2 mmol/L) reduced the levels of apoA-I protein but not mRNA and inhibited apoA-1 protein synthesis in mouse primary hepatocytes. Further, plasma lecithin:cholesterol acyltransferase (LCAT) substrate reactivity was decreased, LCAT specific activity increased, and plasma LCAT protein levels unchanged in apoE−/−/CBS−/− mice. Finally, the clearance of plasma HDL cholesteryl ester, but not HDL protein, was faster in CBS−/−/apoE−/− mice, correlated with increased scavenger receptor B1, and unchanged ATP-binding cassette transporter A1 protein expression in the liver. These findings indicate that HHcy inhibits reverse cholesterol transport by reducing circulating HDL via inhibiting apoA-I protein synthesis and enhancing HDL-C clearance.

Isolation and characterization of the major apolipoprotein from chicken high density lipoproteins

High density lipoproteins were isolated from plasma of white Leghorn hens by ultracentrifugal flotation between densities 1.063 and 1.210 g/ml. After delipidation, the lipid-free proteins were fractionated by chromatography on Sephadex G-150 in urea; one major apolipoprotein was isolated and characterized. From its chemical, physical and immunochemical properties, the major apoprotein from hen high-density lipoproteins has characteristics similar to the major apoprotein of human high density lipoproteins, apoA-I. Thus the hen protein has been designated hen apoA-I. Hen apoA-I has a molecular weight of approximately 28 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Its calculated molecular weight from its 234 constituent amino acids is 26 674. Hen apoA-I differed from its human counterpart by containing isoleucine. Treatment of hen apoA-I with carboxypeptidase A yielded a COOH-terminal sequence of Leu-Val-Ala-Gln. Automatic Edman degradation of the apoprotein gave an NH2-terminal sequence of Asp-Glu-Pro-Gln-Pro-Glu-Leu. Hen apoA-I had a circular dichroic spectrum typical of alpha-helical structures; the calculated helicity was 90%. Goat antisera prepared to hen apoA-I formed precipitin lines of complete identity to the hen apoprotein but lines of only partial identity to human apoA-I. These studies show that the major apoprotein from hen and human high-density lipoproteins have similar properties to each other suggesting a common physiologic function.

A mammalian monothiol glutaredoxin, Grx3, is critical for cell cycle progression during embryogenesis

Glutaredoxins (Grxs) have been shown to be critical in maintaining redox homeostasis in living cells. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified. However, the biological and physiological functions of this group of proteins have not been well characterized. Here, we characterize a mammalian monothiol Grx (Grx3, also termed TXNL2/PICOT) with high similarity to yeast ScGrx3/ScGrx4. In yeast expression assays, mammalian Grx3s were localized to the nuclei and able to rescue growth defects of grx3grx4 cells. Furthermore, Grx3 inhibited iron accumulation in yeast grx3gxr4 cells and suppressed the sensitivity of mutant cells to exogenous oxidants. In mice, Grx3 mRNA was ubiquitously expressed in developing embryos, adult tissues and organs, and was induced during oxidative stress. Mouse embryos absent of Grx3 grew smaller with morphological defects and eventually died at 12.5 days of gestation. Analysis in mouse embryonic fibroblasts revealed that Grx3−/− cells had impaired growth and cell cycle progression at the G2/M phase, whereas the DNA replication during the S phase was not affected by Grx3 deletion. Furthermore, Grx3‐knockdown HeLa cells displayed a significant delay in mitotic exit and had a higher percentage of binucleated cells. Therefore, our findings suggest that the mammalian Grx3 has conserved functions in protecting cells against oxidative stress and deletion of Grx3 in mice causes early embryonic lethality which could be due to defective cell cycle progression during late mitosis.

Chicken apolipoprotein A-I: cDNA sequence, tissue expression and evolution.

Using an antibody against chicken apolipoprotein (apo) A-I, we identified multiple cDNA clones for the protein in two intestinal cDNA libraries in lambda gt11. The complete nucleotide sequence of chicken apoA-I cDNA was determined. The sequence predicts a mature protein of 240 amino acids, a 6-amino acid propeptide and an 18-amino acid signal peptide. Using a 32P-cDNA probe, we detected the presence of apoA-I mRNA in 21 day old chicken intestine, liver, kidney, spleen, breast muscle and brain. The primary sequence of apoA-I contains numerous tandem repeats of 11 and 22 residues in a manner similar to the mammalian proteins. Our analysis of apoA-I sequences from human, rabbit, dog, rat, and chicken indicates that the rate of amino acid substitution is considerably faster in the rat lineage than in other mammalian lineages.

The primary structure of human apolipoprotein A-IV.

Human apolipoprotein (apo) A-IV was purified from chylous ascites fluid. Proteolytic peptides produced by trypsin and Staphylococcus aureus V8 proteinase digestions were purified by high-performance liquid chromatography and sequenced. Human apoA-IV contains 376 amino acid residues. The peptide-derived sequence generally matches two previously reported DNA-derived amino acid sequences except for discrepancies in five positions. In order to examine these discrepancies further, one complete apoA-IV cDNA clone and another partial clone were sequenced. Comparison of all the available information indicates that the peptide-derived sequence reported here is accurate. Sequencing errors probably account for some of the discrepancies between the two primary sequences predicted by earlier nucleotide analyses. In certain positions, however, bona fide sequence heterogeneity or cloning artifact cannot be excluded.

Structure and conformational analysis of lipid-associating peptides of apolipoprotein B-100 produced by trypsinolysis

Apolipoprotein B-100 (apo B-100) contains putative lipid-associating regions that are, in part, responsible for its overall structure in human plasma low-density lipoproteins. Some of these regions have been identified by reassembly of the total tryptic peptides of apo B-100 with bovine brain sphingomyelin, 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and dimyristoylphos-phatidylcholine (DPMC). Although more than 500 tryptic peptides are predicted from the known number of arginines and lysines in apo B-100, significant amounts of only 13 peptides spontaneously associate with all three phospholipids. These peptides share some structural characteristics, as predicted by several algorithms, that distinguish them from the water-soluble apolipoproteins. Most apolipoproteins associate with lipids via amphipathic helices and are highly helical in native and reassembled lipoproteins. Analysis of all apo B-100 lipophilic peptides by circular dichroism and by use of a predictive algorithm reveals no evidence of amphipathic helices. Although the predictive algorithm suggested that the lipophilic peptides of apo B-100 contain the sequence determinants for β-sheet, no spectroscopic evidence for this structure was found. We conclude that the lipophilic regions of apo B-100 liberated by trypsinolysis are highly hydrophobic, although their secondary structures do not fit any simple model.

Cloning and sequencing of bovine apolipoprotein E complementary DNA and molecular evolution of apolipoproteins E, C-I, and C-II

SummaryApolipoprotein (apo) E, a major protein component of plasma lipoproteins, is a physiological ligand for the low density lipoprotein (LDL) receptor as well as for a specific apoE receptor; it is therefore an important modulator of lipoprotein metabolism. In this study we cloned and sequenced bovine apoE complementary DNA. Comparison of nucleotide substitution rates shows that apoE is less conservative than apoA-I and evolves about 30% faster than an average mammalian protein. Although apoE is not a conservative protein, several regions have been well conserved among all eight mammalian sequences now available. These include a 33-amino-acid block immediately upsteam from the third intron/exon junction and the LDL receptor binding region. We have also compared published apoC-I and apoC-II sequences. Both proteins are less conservative than apoE. In particular, apoC-I shows no well-conserved region except for a small region in the common 33-amino-acid block, suggesting that the function of apoC-I does not have stringent structural requirements. On the other hand, in apoC-II the region encoded by exon 4, which consists of the last 29 amino acids of the polypeptide, has been rather well conserved, probably because this region is important for the activation of lipoprotein lipase and chylomicron and very low density lipoprotein metabolism.

Chapter 2 The complete structures of human apolipoprotein B-100 and its messenger RNA

Publisher Summary This chapter highlights the complete structure of human apolipoprotein B (ApoB)-100 and its messenger RNA. ApoB is an important component in the system of plasma lipoproteins. It functions as the ligand for the low-density lipoprotein (LDL) receptor in peripheral cells, and is a component of plasma chylomicrons (CM), very low-density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low-density lipoproteins (LDL). ApoB-100 is synthesized by the liver and is an obligatory constituent of VLDL, IDL, and LDL. Apo-B is insoluble in both 4.2 M tetramethyl urea and aqueous buffers after extraction with organic solvents. The composition of ApoB following partial tryptic digestion and cyanogen bromide cleavage are all similar to one another. Human plasma LDL contains approximately 80% lipid and 20% protein by weight. About 4–10% of the mass of apoB consists of carbohydrate chains containing galactose, mannose, N-acetylglucosamine, and sialic acid residues. ApoB-100 in LDL is highly immunogenic, and high-titer antibodies against human LDL can be consistently produced by injection of LDL into rabbits.

Structural Features that Determine the Functional Properties of the Loop Region of Human Lipoprotein Lipase

Lipoprotein lipase, hepatic triglyceride lipase, and pancreatic lipase have high sequence homology. The crystal structure of human pancreatic lipase and site specific mutagenesis of lipoprotein lipase and hepatic lipase demonstrate an Asp-His-Ser catalytic triad covered by a peptide loop bounded by a conserved disulfide bridge. Substitution of the loop of lipoprotein lipase with either six or twenty-two residues of the hepatic lipase loop gives mutants with complete and partial activity, respectively. Partial or complete substitution with the pancreatic lipase loop produces an inactive protein. Comparison of the primary sequences and the predicted secondary structures identify Glu220and a large hydrophobic moment of the first predicted α-helical region as structural features of lipoprotein lipase that distinguish it from hepatic and pancreatic lipase and may account for the much greater hydrolytic activity of lipoprotein lipase on a triolein substrate.