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.     

Tissue-specific methylation patterns and expression of the human apolipoprotein AI gene.

To better understand the tissue-specific expression of the human apolipoprotein (apo)AI gene, we performed a detailed analysis of the pattern of methylation of the gene in various human adult and embryonic tissues and in tissues of transgenic mice harboring the human apo-AI gene. In addition, the gene was analyzed also in liver and intestine-derived human cell lines (HepG2 and Caco2, respectively). Using methyl-sensitive restriction enzymes (HpaII, HhaI, and SmaI) and the appropriate radioactive probes, we were able to determine separately the status of methylation of the 5'-end, the body of the gene, and 3'-end flanking sequences. The apo-AI gene in tissues that express the gene was undermethylated at the 5'-end. However, the 5'-end of the gene in sperm and in all adult tissues that do not express the gene was heavily methylated. The body of the gene which contains a CpG island and the 3'-end flanking sequences were, in general, hypomethylated except for specific sites that showed partial methylation. In contrast, while the gene showed tissue-specific expression already in a 12-week-old embryo, the 5'-end was invariably hypomethylated in all tissues of the embryo. A human apo-AI transgene has recently been shown to be active exclusively in the liver, while the endogenous gene is expressed in both liver and intestine (6). We show here that the 5'-end of the apo-AI transgene was methylated in all tissues of the mouse (including intestine) except liver. The results presented here demonstrate a clear correlation between hypomethylation of the 5'-end and activity of the apo-AI gene. However, the observed methylation pattern of the gene in embryonic tissues suggests that tissue-specific expression precedes formation of the tissue-specific methylation pattern.     

Apolipoprotein AIMarburg: Studies on two kindreds with a mutant of human apolipoprotein AI

Summary Three probands heterozygous for a mutant of apolipoprotein AI (apo AI_Marburg, Utermann et al. 1982a) were detected by screening of 2282 unrelated individuals resulting an a frequency estimate of about 1/750 in the German population. All three probands with apo AI_Marburg had hypertriglyceridemia (triglyceride above 250 mg/dl) and subnormal HDL-cholesterol (below 30 mg/dl), but no other lipoprotein abnormalities. The kindreds of two probands with AI_Marburg were studied. The family data are consistent with an autosomal codominant inheritance of the trait. A total of 16 heterozygous blood relatives with the mutant AI_Marburg were detected in these kindreds.Analysis of the plasma lipid and lipoprotein levels in relation to the apo AI phenotype was complicated by the high prevalence of diabetes mellitus and thyroid disease in one kindred and of hyperlipidemia in both kindreds. No consistent relationship between plasma lipid and lipoprotein levels, and the mutant apo AI could be demonstrated. Instead the mutant apo AI and the dyslipoproteinemia seem to co-exist independently in these kindreds. Three sibs with the homozygous apo E-2/2 phenotype were detected in one kindred, and all three sibs had subnormal LDL-cholesterol and beta-VLDL, e.g., the lipoprotein abnormality characterizing primary dysbetalipoproteinemia. Genetic apo E phenotypes and the apo AI mutant segregated independently, indicating that the structural gene loci for apo E and apo AI are not closely linked.     

Functional characterization of Asp-317 mutant of human renin expressed in COS cells

Renin is an unique aspartyl (acid) protease with optimal activity at neutral pH. It has been suggested that Ala-317 of human renin contributes to neutral optimum pH of the enzyme [(1984) FEBS Lett. 174, 102–111]. The hypothesis was verified by the characterization of mutant renin in which Ala-317 was replaced with Asp by a site-directed mutagenesis. Wild-type and mutant renins, which were expressed in COS cells, exhibited different pH-activity profiles and optimum pH of the mutant enzyme was lower than that of the wild-type enzyme. This result suggests that Ala-317 of human renin plays an important role in the determination of optimum pH of the enzyme.     

Nuclear localisation of wild type and mutant galectin-3 in transfected cells

Galectin-3, a member of a family of carbohydrate-binding proteins, is present generally in the cytoplasm of cells. However, galectin 3 can also be located in nuclei under certain conditions although it lacks any known nuclear localisation signal and the mechanism by which the protein is sequestered in nuclei is unknown. Here we describe that Cos-7 cells or rabbit smooth muscle Rb-1 cells transfected with cDNA encoding hamster galectin-3 sequester the protein in nuclei whereas untransfected BHK cells expressing the endogenous hamster lectin or transfected BHK cells over-expressing the protein, do not. Confocal immunofluorescence microscopy of Cos-7 cells or rabbit smooth muscle Rb-1 cells transfected with cDNAs encoding mutants of hamster galectin-3 containing N-terminal or internal deletions shows that nuclear localisation does not require the first 103 amino acid residues of the protein. Further deletion of residues 104-110 dramatically prevents sequestration in nuclei. However, the sequence A104PTGALT110 by itself is not obligatory for nuclear localisation and can be substituted by other unrelated sequences. A truncated galectin-3 protein, that is blocked in nuclear expression, retains carbohydrate-binding activity, making less likely the possibility that severe N-terminal truncations of galectin-3 induce mis-folding leading to aggregation and cytoplasmic sequestration and an incidental effect on nuclear trafficking. These studies indicate that nuclear import and retention of galectin-3 is a property of the CRD domain and is independent of N-terminal domains that others have shown to contain binding domains for various nuclear components.     

A potential mucus precursor in Tetrahymena wild type and mutant cells.

By using an antibody to a specific mucus polypeptide (34 kDa) to study whole cell extracts of both a secretory mutant (SB281) and wild type (wt) Tetrahymena, we demonstrate that a 57-kDa polypeptide is a probable precursor to the 34-kDa secretory polypeptide. We postulate that the precursor accumulates in the mutant cells because it cannot be cleaved. This mutant contains no recognizable mature secretory granules (mucocysts). By immunoelectron microscopy, the 34-kDa polypeptide was localized in wt cells specifically to the mature mucocysts and to their released products. Localization in mutant cells occurred in two different types of cytoplasmic vesicles: small electron dense vesicles (0.3-0.5 microns in diameter) and large electron lucent vacuoles (1.2-3.5 microns in diameter). Immunoblot analyses of homogenates of mutant and wt cells with the anti-34-kDa serum revealed a dominant band in the mutant at Mr 57 kDa whereas the wt showed a dominant band only at Mr 34 kDa. Furthermore, the 57-kDa polypeptide is immunoprecipitated with anti-34-kDa serum from the mutant cell. Further evidence for a precursor relation of the 57-kDa polypeptide in mutant cells to the 34-kDa mucus polypeptide of wt cells was obtained by the use of drugs (monensin, chloroquine, NH4Cl) that block secretory product processing in wt cells. Extracts of drug-treated wt cells showed the presence of a 57-kDa cross reacting band even after 18 h of incubation in growth medium whereas untreated control cells contained the 34-kDa mature protein almost exclusively. These results indicate that processing of the precursor to the 34-kDa polypeptide occurs in an acidic compartment(s) possibly in either the trans Golgi network, or condensing vacuoles or both.     

Differential cytotoxicity of human wild type and mutant alpha-synuclein in human neuroblastoma SH-SY5Y cells in the presence of dopamine

Parkinson's disease (PD) involves loss of dopaminergic neurons in the substantia nigra and is characterized by intracellular inclusions, Lewy bodies, consisting primarily of aggregated alpha-synuclein. Two substitution mutations (A53T and A30P) in alpha-synuclein gene have been identified in familial early-onset PD. To understand the biological changes that incur upon alpha-synuclein-induced cytotoxicity in the presence of dopamine, the current studies were undertaken. Human SH-SY5Y neuroblastoma cells coexpressing the human dopamine transporter [hDAT], and either wild type (wt) or mutant alpha-synucleins, were treated with 50 microM dopamine (DA). In cells expressing wt or A30P alpha-synuclein, DA accelerated production of reactive oxygen species and cell death as compared to cells expressing A53T or hDAT alone. The increased sensitivity of such cells to DA was investigated by measuring changes in cellular ionic gradient, by atomic absorption spectrometry, and cell metabolism, by high-resolution nuclear magnetic resonance spectroscopy. Both wt and A30P alpha-synuclein caused rapid decrease in levels of intracellular potassium, followed by mitochondrial damage and cytochrome c leakage, with decreased cellular metabolism as compared to cells expressing A53T or hDAT alone. Collapse of ionic gradient was significantly faster in A30P (t(1/2) = 3.5 h) than in wt (t(1/2) = 6.5 h) cells, and these changes in ionic gradient preceded cytochrome c leakage and depletion of metabolic energy. Neither wt nor mutant alpha-synuclein resulted in significant changes in ionic gradient or cellular metabolism in the absence of intracellular DA. These findings suggest a specific sequence of events triggered by dopamine and differentially exacerbated by alpha-synuclein and the A30P mutant.     

Effectivity of expression of mature forms of mutant human apolipoprotein A-I.

In order to probe the structural and functional properties of a central region of apolipoprotein A-I (apoA-I), we engineered mutants of the mature form of the protein and expressed them using the baculovirus/insect cell expression system. The mutations which targeted the region of apoA-I between amino acids 140 and 150 included: (i) deletion of the region 140-150 (apoA-I(Delta140-150)); (ii) substitution of arginine 149 with valine (apoA-I(R149V)); (iii) substitution of proline 143 with alanine (apoA-I(P143A)); (iv) deletion of region 63-73 (apoA-I(Delta63-73)), which has structural properties similar to 140-150; and (v) a chimeric protein substituting amino acids 140-150 with amino acids 63-73 (apoA-I(140-150 --> 63-73)). The efficiencies of synthesis were vastly different for the various mutants as follows: apoA-I(R149V) > apoA-I(140-150 --> 63-73) > apoA-I(Delta63-73) > apoA-I(P143A) > apoA-I > apoA-I(Delta140-150). About 50% of the synthesized wild type and all apoA-I mutants was retained in the cells. During expression of apoA-I(R149V) an unusual spontaneous recombination occurred. In addition to the expected mutant, another form of apoA-I with an apparent M(r) of 36K was produced which consisted of a duplication of the amino-terminal end of apoA-I, from the prepeptide through to amino acid 62, linked to the original pre-apoA-I(R149V) sequence via a 4-amino-acid linker. Despite the fact that this form of apoA-I carries two prepeptides and consequently two cleavage sites, there was little, if any, cleavage at the internal cleavage site. During expression, less than 20% of this mutant was retained in the cells. These results demonstrate that at least in the model of insect cells, the efficiency of apoA-I synthesis, processing, and secretion depends on apoA-I secondary structure and/or folding.     

Heterologous expression and ATPase activity of mutant versus wild type PfMDR1 protein

Mutation of the P. falciparum chloroquine resistance transporter (PfCRT) causes resistance to chloroquine (CQ) and other antimalarial drugs. Mutation and/or overexpression of one of the multidrug resistance protein homologues found in this malarial parasite (PfMDR1) may further modify or tailor the degree of multidrug resistance. However, considerable controversy surrounds the precise contribution of PfMDR1, in part because no direct biochemical studies of PfMDR1 have yet been possible. Using codon optimization and other principles, we have designed and constructed a yeast optimized version of the wild type pfmdr1 gene and have successfully overexpressed PfMDR1 protein in P. pastoris yeast. The protein is well expressed in either full length form or as two separate half transporters, is well localized to the yeast plasma membrane and is fully functional as evidenced by ATPase activity measurements. We have also expressed mutants that have previously been hypothesized to influence drug resistance in parasites. Using purified plasma membrane fractions, we have analyzed antimalarial drug effects on ATPase activity for wild type versus mutant proteins. Relative to other ABCB transporters involved in drug resistance, PfMDR1 is unusual. It has similar pH, [ATP], and Mg++ dependencies for ATP hydrolysis, yet relatively high Km and Vmax values for ATP hydrolysis, and ATPase activity is only mildly stimulated by antimalarial drugs. The largest measured drug effect is for CQ (to which PfMDR1 is not believed to confer resistance), and it is strongly inhibitory for WT PfMDR1. Drug resistance associated PfMDR1 mutants show either elevated (Dd2 allele encoded) or reduced (7G8 allele) basal ATPase activity and different patterns of drug stimulation or inhibition, relative to WT PfMDR1. The Dd2 PfMDR1 isoform also shows a slightly more alkaline pH optimum. Surprisingly, verapamil alone (1-300 microM) does not significantly affect WT ATPase activity but inhibits the Dd2 isoform at 1 microM. These data should assist ongoing analysis of the contribution of PfMDR1 to antimalarial drug resistance.     

人溶酶体酸性b-葡萄糖脑苷脂酶基因的克隆及其在COS7细胞中的表达

从人胎盘组织提取总RNA, 采用RT-PCR扩增人溶酶体酸性b-葡萄糖脑苷脂酶(Lysosomal acid b-glucosidase, GlcCerase)基因编码区的全部序列, 并克隆到pMD-19T载体上, 构建克隆载体pMD-GlcCerase。经测序验证后, 将GlcCerase亚克隆至表达载体pEGFP-C1上, 构建了人GlcCerase绿色荧光蛋白真核表达载体pEGFP-GlcCerase。采用脂质体法将其瞬时转染至COS7细胞系后, 在细胞中检测到了GlcCerase基因, 并在细胞裂解产物中检测到了GlcCerase生物活性的表达。GlcCerase基因的克隆及其表达, 为进一步了解GlcCerase基因的功能以及利用转基因动物乳腺生物反应器高效生产GlcCerase奠定了基础。     

High yield and secretion of recombinant human apolipoprotein AI in Pichia pastoris

Apolipoprotein AI (ApoAI) is an important apolipoprotein in plasma and is known to have various physiological functions suitable for pharmaceutical applications. Human blood has been the only source of this protein for research and large-scale applications. To obtain large amounts of ApoAI a Pichia pastoris expression system was first used to obtain a high level of expression of secreted, recombinant protein. The human gene encoding ApoAI was inserted into the secretion vector pPIC9K and used to transform P. pastoris GS115. AP16, a high expression transformant with high G418 resistance, was obtained. After induction with methanol, the expression level of rhApoAI (recombinant human ApoAI) was 160 mg/L in a 14L fermentor. RhApoAI was purified by cold acetone precipitation followed by Q-Sepharose Fast Flow ion exchange column chromatography with 60% recovery. The N-terminal amino acid sequence and molecular weight (mass spec.) of rhApoAI are identical to native human ApoAI. Purified rhApoAI has specific binding activity with liver cells SMC7721 and binding can be inhibited by native human ApoAI.     

Differential expression of apolipoprotein d in human astroglial and oligodendroglial cells.

Apolipoprotein D (Apo D) is a secreted lipocalin in the nervous system that may be related to processes of reinnervation and regeneration. Under normal conditions, Apo D is present in the central nervous system in oligodendrocytes, astrocytes, and some scattered neurons. To elucidate the regional and cellular distribution of Apo D in normal human brain, we performed double immunohistochemistry for glial fibrillary acidic protein (GFAP) and Apo D in samples of postmortem human cerebral and cerebellar cortices. Most of the GFAP-positive cells in the gray matter had features of protoplasmic astrocytes and were mainly Apo D-positive. Apo D staining was mostly confined to the cell soma and proximal processes, whereas GFAP extended to a rich and extensive array of processes. The fibrous astrocytes in the white matter were immunoreactive for GFAP but not for Apo D. In the white matter, Apo D was mainly detected in oligodendrocytes and extracellularly in the neuropil. The results of the present study support a specific behavior for each astrocyte type. These findings suggest that Apo D expression may be cell-specific, depending on the particular tissue physiology at the time of examination.     

Role of interferon in the replication of human parainfluenza virus type 1 wild type and mutant viruses in human ciliated airway epithelium

Human parainfluenza virus type 1 (HPIV1) is a significant cause of pediatric respiratory disease in the upper and lower airways. An in vitro model of human ciliated airway epithelium (HAE), a useful tool for studying respiratory virus-host interactions, was used in this study to show that HPIV1 selectively infects ciliated cells within the HAE and that progeny virus is released from the apical surface with little apparent gross cytopathology. In HAE, type I interferon (IFN) is induced following infection with an HPIV1 mutant expressing defective C proteins with an F170S amino acid substitution, rHPIV1-C^F170S, but not following infection with wild-type HPIV1. IFN induction coincided with a 100- to 1,000-fold reduction in virus titer, supporting the hypothesis that the HPIV1 C proteins are critical for the inhibition of the innate immune response. Two recently characterized live attenuated HPIV1 vaccine candidates expressing mutant C proteins were also evaluated in HAE. The vaccine candidates, rHPIV1-C^R84G/Δ170HN^T553AL^Y942A and rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710-11, which contain temperature-sensitive (ts) attenuating (att) and non-ts att mutations, were highly restricted in growth in HAE at permissive (32°C) and restrictive (37°C) temperatures. The viruses grew slightly better at 37°C than at 32°C, and rHPIV1-C^R84G/Δ170HN^T553AL^Y942A was less attenuated than rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710-11. The level of replication in HAE correlated with that previously observed for African green monkeys, suggesting that the HAE model has potential as a tool for the preclinical evaluation of HPIV1 vaccines, although how these in vitro data will correlate with vaccine virus replication in seronegative human subjects remains to be seen.     

Gene expression in human cells with mutant insulin receptors

Insulin initiates its action by interacting with specific receptors on the plasma membrane of target cells. Mutations in these receptors cause the inherited insulin-resistant syndrome leprechaunism. Affected patients have severe intrauterine and post-natal growth restriction coupled with severe metabolic abnormalities. Fibroblasts from patients with leprechaunism have impaired in vitro growth, reflecting the growth restriction seen it in vivo. To determine the reason for the defective growth of cells from patients with mutant insulin receptors, gene expression was compared among fibroblasts from controls and patients with leprechaunism using DNA microarrays. Of the 12,626 human genes tested, cells from patients with leprechaunism had consistently increased mRNA for 151 genes and decreased mRNA for 51 genes. The level of expression of selected genes was independently confirmed by real time RT-PCR. Leprechaun cells had increased expression of several genes involved in metabolic functions, several of which were not previously known to be regulated by the insulin receptor. The absence of insulin receptors modified the expression of genes controlling apoptosis and cellular growth. Functional analysis indicated that cells from patients with leprechaunism had a normal response to apoptotic stimuli when mitochondrial potential and caspase activity were assayed. About 20% of the genes whose RNA was decreased in leprechaun cells coded for proteins involved in cell growth and differentiation. These results suggest that the insulin receptor is a physiologic regulator of several genes involved in intermediate metabolism even in human fibroblasts. Decreased expression of growth-promoting genes may explain the growth restriction of patients with severe insulin resistance.