Mutagenesis of the glycosylation site of human ApoCIII. O-linked glycosylation is not required for ApoCIII secretion and lipid binding

We have used site-directed in vitro mutagenesis to alter the codon ACT of human apoCIII gene, specifying Thr-74, to GCT (Ala-74). The normal and mutant apoCIII genes were then placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus vector and were used for cell transfection and selection of stable cell lines. Blotting analysis of RNA isolated from several independent cell clones showed that both the normal and mutant genes produced apoCIII mRNA in amounts larger than that found in human fetal liver. Pulse-chase analysis of cell clones expressing the normal and mutant apoCIII genes showed that only the normal apoCIII is modified intracellularly to produce a disialated form (apoCIIIs2). Cell clones expressing the normal apoCIII gene secrete exclusively the disialated form, whereas those expressing the mutant gene secrete the unmodified form. The amount of mutant apoCIII protein produced by C127 cell clones expressing the mutant gene was reduced as compared to that produced by the control cells. Density gradient ultracentrifugation analysis of the secreted apoCIII showed that the flotation properties of the secreted normal and mutant proteins were similar. These findings suggest that the intracellular glycosylation of apoCIII is not required for its intracellular transport and secretion. Furthermore, lack of glycosylation has no effect on the relative affinities of apoCIII for plasma very low density lipoproteins and high density lipoproteins.     

Expression and secretion of chicken apolipoprotein AI in transfected COS cells

A full-length chicken apolipoprotein A-I (apoAI) cDNA has been cloned into an expression vector, pRSVapoAI. This plasmid was transfected into a monkey kidney (COS-1) cell line in order to study apolipoprotein-lipid assembly. Chicken apoAI is the major apolipoprotein of chicken high-density lipoprotein (HDL), which is less complex in apolipoprotein content than the HDL of human plasma. The transient transfected COS-1 cells synthesized and secreted authentic plasma apoAI. Under serum-free medium conditions, COS cells secreted only proapoAI. A small portion (15%) of the secreted apoAI floated at a density 1.07-1.20 g/ml. Upon incubation with fetal bovine serum at 10 degrees C, a majority of the apoAI was recovered in the HDL density (1.06-1.20 g/ml) region. Secreted apoAI was labeled when transfected COS cells were incubated with [U-14C]palmitate, but the incorporation of radioactivity was not the result of fatty acid acylation through ester bond formation. These results indicate that heterologous COS-1 cells are capable of synthesizing and secreting apoAI, and that intracellular association of apoAI with lipids is not necessary for secretion.     

Estrogen upregulates hepatic apolipoprotein M expression via the estrogen receptor

Apolipoprotein M (apoM) is present predominantly in high-density lipoprotein (HDL) in human plasma, thus possibly involved in the regulation of HDL metabolism and the process of atherosclerosis. Although estrogen replacement therapy increases serum levels of apoAI and HDL, it does not seem to reduce the cardiovascular risk in postmenopausal women. Therefore, we investigated the effects of estrogen on apoM expression in vitro and in vivo. HepG2 cells were incubated with different concentrations of estrogen with or without the estrogen receptor antagonist, fulvestrant, and apoM expression in the cells was determined. Hepatic apoM expression and serum levels of apoM were also determined in normal and in ovariectomized rats treated with either placebo or estradiol benzoate, using sham operated rats as controls. Estrogen significantly increased mRNA levels of apoM and apoAI in HepG2 cell cultures in a dose- and time-dependent manner; the upregulation of both apolipoproteins was fully abolished by addition of estrogen receptor antagonist. In normal rats, estrogen treatment led to an increase in plasma lipid levels including HDL cholesterol, a marked upregulation of apoM mRNA and a significant increase in serum levels of apoM. The same pattern of regulation was found in ovariectomized rats treated with estrogen. Thus, estrogen upregulates apoM expression both in vivo and in vitro by mechanism(s) involving the estrogen receptor.     

Human metallothionein genes: molecular cloning and sequence analysis of the mRNA.

From a cDNA clone bank prepared from cadmium-treated HeLa cells, we isolated clones representing mRNAs whose concentration is increased after cadmium induction. Several metallothionein cDNA clones were isolated by cross-hybridization to mouse metallothionein-I cDNA. The nucleotide sequence of one of these clones, containing a nearly full-length cDNA copy of human metallothionein-II mRNA, was determined. The homology between the human and mouse metallothionein sequences is strictly limited to the coding region of the mRNA. Codon usage in metallothionein mRNA is not random. Seventy-nine percent of the codons have G or C residues at the third position, resulting in a GC-rich sequence.     

Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.     

A complete NMR spectral assignment of the lipid-free mouse apolipoprotein A-I (apoAI) C-terminal truncation mutant, apoAI(1-216)

ApoAI is the major protein component of the high-density lipoprotein (HDL) that has been a hot subject of interests because of its anti-atherogenic properties. Lipid-free apoAI specifically binds to phospholipids, triggering HDL formation. Here we report a complete backbone assignment and nearly complete sidechain assignment of a C-terminal 24-residue truncation mutant of mouse apoAI, apoAI(1-216), in its lipid-free form.     

Stably transfected ABCA1 antisense cell line has decreased ABCA1 mRNA and cAMP-induced cholesterol efflux to apolipoprotein AI and HDL.

Using a sensitive real time fluorescent PCR assay, ABCA1 mRNA levels were induced by approximately 50-70-fold following 8Br-cAMP treatment of the RAW264 murine macrophage cell line, concomitant with the induction of cholesterol efflux to apoAI and HDL. A stably transfected ABCA1 antisense cDNA cell line was created, which led to approximately 50-70% reductions in ABCA1 mRNA levels in basal and 8Br-cAMP-treated cells, and diminished to the same extent the 8Br-cAMP-mediated efflux of cholesterol to apolipoprotein AI and HDL. These data demonstrate that ABCA1 is necessary for the cAMP-induced lipid efflux to both apoAI and HDL.     

Secretion of apolipoprotein A-I in lipoprotein particles following transfection of the human apolipoprotein A-I gene into 3T3 cells

Apolipoprotein A-I (apoA-I) is the major protein constituent of plasma high density lipoproteins (HDL). To examine apoA-I processing and secretion, the human apoA-I gene (2.2-kilobase PstI-PstI fragment) linked to the mouse metallothionein promoter was transfected by electroporation into NIH 3T3 fibroblasts along with the plasmid pSV2 neo, which confers neomycin resistance. Transfected cells were selected for neomycin resistance and screened for the ability to produce apoA-I by enzyme-linked immunosorbent assay. In the absence of lipids in the medium, selected 3T3 cells secreted apoA-I, mainly in the proprotein form, at density greater than 1.25 g/ml. Following incubation of cells with lipids, and subsequent washing with lipid-free medium, apoA-I was recovered in the HDL region (1.063-1.21 g/ml) as well as in the 1.21 g/ml infranatant. Examination of the HDL fraction by electron microscopy revealed round particles, 10-21 nm in diameter. These data indicate that human apoA-I secreted by transfected 3T3 fibroblasts can assemble into lipoprotein particles under the appropriate conditions.     

Analysis of human monoclonal antibodies derived from lymphocytes of patients with cancer

Human lymphocytes from tumor-bearing patients and normal individuals have been fused with the NS-1 mouse myeloma line or the LICR -LON- HMY2 ( LICR -2) or SK0 -007 human cell lines. For a given number of lymphocytes, fusions with NS-1 produced 8 times more clones than fusions with LICR -2 and greater than 20 times more clones than fusions with SK0 -007. The percentage of clones that secrete human immunoglobulin (Ig) and the range of Ig production were comparable for clones derived from the three myeloma/lymphoblastoid lines. Clones derived from fusions with LICR -2 and SK0 -007 were found to secrete new species of light and heavy Ig chains in addition to those of the myeloma/lymphoblastoid lines, and clones derived from fusions with NS-1 secreted human Ig and contained both mouse and human chromosomes, which indicates that true hybrid cells were derived from fusions with each of the myeloma/lymphoblastoid lines under study. The stability of Ig production was similar for clones derived from fusions with NS-1, LICR -2, or SK0 -007; these results were comparable to those obtained with standard mouse/mouse hybrids. Stable clones producing human monoclonal antibodies that react with cell surface, cytoplasmic, cytoskeletal, nuclear, or nucleolar antigens have been isolated from tumor-bearing patients and normal individuals. A number of human monoclonal antibodies reactive with cytoskeletal antigens appear to be directed against components of the intermediate filament family. Techniques for the production of human monoclonal antibody appear to be sufficiently advanced to initiate a serological dissection of the humoral immune response to cancer.     

Induction of the phospholipid transfer protein gene accounts for the high density lipoprotein enlargement in mice treated with fenofibrate

Fibrate treatment in mice is known to modulate high density lipoprotein (HDL) metabolism by regulating apolipoprotein (apo)AI and apoAII gene expression. In addition to alterations in plasma HDL levels, fibrates induce the emergence of large, cholesteryl ester-rich HDL in treated transgenic mice expressing human apoAI (HuAITg). The mechanisms of these changes may not be restricted to the modulation of apolipoprotein gene expression, and the aim of the present study was to determine whether the expression of factors known to affect HDL metabolism (i.e. phospholipid transfer protein (PLTP), lecithin:cholesterol acyltransferase, and hepatic lipase) are modified in fenofibrate-treated mice. Significant rises in plasma PLTP activity were observed after 2 weeks of fenofibrate treatment in both wild-type and HuAITg mice. Simultaneously, hepatic PLTP mRNA levels increased in a dose-dependent fashion. In contrast to PLTP, lecithin:cholesterol acyltransferase mRNA levels in HuAITg mice were not significantly modified by fenofibrate despite a significant decrease in plasma cholesterol esterification activity. Fenofibrate did not induce any change in hepatic lipase activity. Fenofibrate significantly increased HDL size, an effect that was more pronounced in HuAITg mice than in wild-type mice. This effect in wild-type mice was completely abolished in PLTP-deficient mice. Finally, fenofibrate treatment did not influence PLTP activity or hepatic mRNA in peroxisome proliferator-activated receptor-alpha-deficient mice. It is concluded that 1) fenofibrate treatment increases plasma phospholipid transfer activity as the result of up-regulation of PLTP gene expression through a peroxisome proliferator-activated receptor-alpha-dependent mechanism, and 2) increased plasma PLTP levels account for the marked enlargement of HDL in fenofibrate-treated mice.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

Expression of the hepatitis B virus surface antigen in mammalian cells using an Epstein-Barr-virus-derived vector

 The hepatitis B virus surface antigen (HBsAg) gene, under control of the inducible mouse metallothionein I gene promoter, was inserted in an expression vector based on the Epstein-Barr virus (EBV). This vector was introduced into human cells by DNA transfection and clones were selected for their resistance to hygromycin B. The recombinant EBV vector replicates efficiently as an episome in human cells and approximately six copies per cell were found in one clone of hygromycin-B-resistant cells. These cells produce high levels of HBsAg in the presence of metals. The protein is mainly found in the cell medium, suggesting that the HBsAg is secreted from the cells. Received: 25 February 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996     

Construction of an EBV-derived shuttle vector for studying the influence of transcription on mutagenesis

We have constructed an EBV-derived shuttle vector, pF1-EBV, which replicates in human cells as an extrachromosomal element. The structural sequences of the gene encoding the bacterial xanthine-guanine-phosphoribosyltransferase (gpt) were fused to the promoter and presumptive control region of the mouse metallothionein I (MT-I) gene. Human 293 cells transformed with the recombinant plasmid synthesized gpt mRNA and the expression of the gene was inducible by zinc. The gpt gene offers a convenient system of selection for mutant plasmids by transformation into the appropriate gpt- E. coli strain. A clonal cell line created by establishment of the pF1-EBV shuttle vector showed a spontaneous gpt- frequency of 2.10(-5). An increase in mutation frequency above background was induced by mutagenizing this cell line with the alkylating agent N-methyl-N-nitrosourea (MNU). The recombinant molecule that we have constructed should provide a tool for studying the role of gene expression in DNA repair and mutagenesis.     

Identification of a human protein homologous to the mouse Lyb-2 B cell differentiation antigen and sequence of the corresponding cDNA

We have isolated and sequenced cDNA clones encoding the human homolog of the mouse Lyb-2 B cell differentiation Ag. Previous data suggest that Lyb-2 might represent a growth factor or lymphokine receptor. Human Lyb-2 mRNA is expressed in normal human tonsils and bone marrow cells, in the pre-B cell line REH, in three Burkitt lymphoma cell lines, and in some EBV-transformed B cell lines, but not in antibody-secreting myeloma cell lines, T cell lines, or a promyelocytic leukemia cell line. These data indicate that expression of human Lyb-2 is restricted to B lineage cells and turned off in antibody-secreting plasma cells. A polyclonal mouse antiserum was raised against human Lyb-2 and immunoprecipitates a Mr 42,000 protein from REH, Raji, and Daudi cells and from mouse L(tk) cells transfected with the human Lyb-2 cDNA in an expression vector. The human Lyb-2 protein is related to both the asialoglycoprotein receptor and CD23, the B cell-specific FcR for IgE. These data demonstrate that human B cells express a previously undescribed cell surface protein that is homologous to mouse Lyb-2 and has a similar pattern of expression during B cell development.     

Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both alpha-LpA-I and prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V(max) (8.4 vs. 8.2 nmol cholesteryl ester/h/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways.     

In vivo studies of HDL assembly and metabolism using adenovirus-mediated transfer of ApoA-I mutants in ApoA-I-deficient mice.

We have used adenovirus-mediated gene transfer in apoA-I-deficient (A-I-/-) mice to probe the in vivo assembly and metabolism of HDL using apoA-I variants, focusing primarily on the role of the C-terminal 32 amino acids (helices 9-10). Lipid, lipoprotein, and apoA-I analyses showed that plasma levels of apoA-I and HDL of the mutants were 40-88% lower than that of wild type (WT) human apoA-I despite comparable levels of expression in the liver. WT apoA-I and mutant 1 (P165A, E172A) formed spherical particles with the size and density of HDL2 and HDL3. Mutant 2 (E234A, E235A, K238A, K239A) generated spherical particles with density between HDL2 and HDL3. Mutant 3 (L211V, L214V, L218V, L219V) and mutant 4 (L222K, F225K, F229K), which have substitutions of hydrophobic residues in the C-terminus, generated discoidal HDL particles indicating a defect in their conversion to mature spherical HDL. Significant amounts of mutant 4 and mutant 5 (truncated at residue 219) were found in the lipid poor fractions after ultracentrifugation of the plasma (18 and 35%, respectively, of total apoA-I). These findings suggest that hydrophobic residues in and/or between helices 9 and 10 are important for the maturation of HDL in vivo.