Characterization and expression of the cDNA encoding a new kind of phospholipid transfer protein, the phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence of a putative membrane targeted phospholipid transfer protein in fungi

The full-length cDNA of a phospholipid transfer protein (PLTP) was isolated from Aspergillus oryzae by a RACE-PCR procedure using degenerated primer pool selected from the N-terminal sequence of the purified phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP). The cDNA encodes a 173 amino acid protein of 18823 Da. The deduced amino acid sequence from position 38 to 67 is 100% identical to the N-terminal sequence (first 30 amino acids) of the purified PG/PI-TP. This amino acid sequence is preceded by a leader peptide of 37 amino acids which is predicted to be composed of a signal peptide of 21 amino acids followed by an extra-sequence of 16 amino acids, or a membrane anchor protein signal (amino acid 5-29). This strongly suggests that the PG/PI-TP is a targeted protein. The deduced mature protein is 138 amino acids long with a predicted molecular mass of 14933 Da. Comparison of the deduced PG/PI-TP sequence with other polypeptide sequences available in databases revealed a homology with a protein deduced from an open reading frame coding for an unknown protein in Saccharomyces cerevisiae (36% identity and 57% similarity). Apart from this homology, the PG/PI-TP is unique and specific to the filamentous fungi on the basis of comparison of PLTP protein sequences. Northern blot analysis of RNA isolated from A. oryzae cultures grown on glucose or glucose supplemented with phospholipids suggests that the PG/PI-TP is transcribed by only one RNA species and allows us to show that expression of the protein is regulated at the messenger RNA level.     

Impact of Aspergillus oryzae genomics on industrial production of metabolites

Aspergillus oryzae is used extensively for the production of the traditional Japanese fermented foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste). In recent years, recombinant DNA technology has been used to enhance industrial enzyme production by A. oryzae. Recently completed genomic studies using expressed sequence tag (EST) analyses and whole-genome sequencing are quickly expanding the industrial potential of the fungus in biotechnology. Genes that have been newly discovered through genome research can be used for the production of novel valuable enzymes and chemicals, and are important for designing new industrial processes. This article describes recent progress of A . oryzae genomics and its impact on industrial production of enzymes, metabolites, and bioprocesses.     

Lipid fatty acid composition of fungi in the genus Aspergillus grown on media with various sources of nitrogen

The fatty acid composition of lipids synthesized by fungi belonging to the Aspergillus genus was studied during their growth on media containing different organic and inorganic nitrogen sources. On the average, the cultures were shown to accumulate from 7 to 30 g/L of biomass and to synthesize from 3 to 13% of lipids. The lipids were found to contain saturated and unsaturated fatty acids with the number of carbon atoms from 14 to 18. The fungi had a typical fatty acid composition of lipids which did not depend on the composition of the growth medium.     

Increased risk of atherosclerosis by elevated plasma levels of phospholipid transfer protein

Plasma phospholipid transfer protein (PLTP) is thought to be involved in the remodeling of high density lipoproteins (HDL), which are atheroprotective. It is also involved in the metabolism of very low density lipoproteins (VLDL). Hence, PLTP is thought to be an important factor in lipoprotein metabolism and the development of atherosclerosis. We have overexpressed PLTP in mice heterozygous for the low density lipoprotein (LDL) receptor, a model for atherosclerosis. We show that increased PLTP activity results in a dose-dependent decrease in HDL, and a moderate stimulation of VLDL secretion (相似文献   

Increased saturated phospholipid in cultured cells grown with linoleic acid

Summary We found that fetal bovine serum supplementation of culture medium provided limited quantities of linoleic acid, an essential fatty acid, to cells grown in culture (2.8 ± 0.3% of total fatty acids in 12 lots). Supplementation of the medium with additional linoleic acid resulted in altered phospholipid acyl composition in cells of two established lines, A549, a putative model of the pulmonary Type II epithelial cell, and SIRC, a line derived from rabbit corneal epithelium. In particular, linoleic acid supplementation induced a relative increase in disaturated choline phosphoglycerides of 33 and 36%, respectively, in cells of the two lines. This observation may be relevant to design of media for primary culture of Type II cells, in which disaturated phospholipid synthesis is used as an index of differentiated function (surfactant production). Linoleate supplementation did not alter growth or size (protein content) of cells of either line and caused a slight increase in accumulation of neutral lipid, in the form of cytoplasmic droplets, in A549 cells. Supplementation of cell cultures with equivalent concentrations of the nonessential fatty acids palmitic and oleic acid did not significantly alter the growth, morphologic appearance, or lipid composition of the cells. However, it was demonstrated in cells of one line that palmitic acid supplementation temporarily stimulated synthesis of disaturated choline phosphoglyceride from radiolabeled choline. This work was supported by Grants HL-24817 and HL-21251 from the National Institutes of Health, USPHS, and by a grant from the Alexandrine and Alexander L. Sinsheimer Fund.     

Determination of human plasma phospholipid transfer protein mass and activity

Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. PLTP is an 80-kDa glycoprotein that is expressed/secreted by a wide variety of tissues including lung, liver, adipose tissue, brain, and muscle. PLTP mediates a net transfer of phospholipids between vesicles and plasma HDLs. It also generates from small HDL particles large fused HDL particles with a concomitant formation of small lipid-poor apolipoprotein (apo) A-I-containing particles which are thought to act as the primary acceptors of cell-derived cholesterol from peripheral tissue macrophages. Another important function of PLTP is connected to lipolysis. Its role in the transfer of surface remnants from triglyceride-rich particles, very-low-density lipoproteins, and chylomicrons, to HDL is of importance for the maintenance of HDL levels. Recent observations from our laboratory have demonstrated that in circulation two forms of PLTP are present, one catalytically active (high-activity form, HA-PLTP) and the other a low-activity form (LA-PLTP). In view of the likely relevancy of PLTP in human health and disease, reliable and accurate methods for measuring plasma/serum PLTP activity and concentration are required. In this chapter, two radiometric PLTP activity assays are described: (i) exogenous, lipoprotein-independent phospholipid transfer assay and (ii) endogenous, lipoprotein-dependent phospholipid transfer assay. In addition, an ELISA method for quantitation of serum/plasma total PLTP mass as well as HA-PLTP and LA-PLTP mass is reported in detail.     

Expression of Aspergillus hemoglobin domain activities in Aspergillus oryzae grown on solid substrates improves growth rate and enzyme production

DNA fragments coding for hemoglobin domains (HBD) were isolated from Aspergillus oryzae and Aspergillus niger. The HBD activities were expressed in A. oryzae by introduction of HBD gene fragments under the control of the promoter of the constitutively expressed gpdA gene. In the transformants, oxygen uptake was significantly higher, and during growth on solid substrates the developed biomass was at least 1.3 times higher than that of the untransformed wild-type strain. Growth rate of the HBD-activity-producing strains was also significantly higher compared to the wild type. During growth on solid cereal substrates, the amylase and protease activities in the extracts of the HBD-activity-producing strains were 30-150% higher and glucoamylase activities were at least 9 times higher compared to the wild-type strain. These results suggest that the Aspergillus HBD-encoding gene can be used in a self-cloning strategy to improve biomass yield and protein production of Aspergillus species.     

Modulation of phospholipid transfer protein activity. Inhibition by local anesthetics

The transfer of phospholipid molecules between biological and synthetic membranes is facilitated by the presence of soluble catalytic proteins, such as those isolated from bovine brain which interacts with phosphatidylinositol and phosphatidylcholine and from bovine liver which is specific for phosphatidylcholine. A series of tertiary amine local anesthetics decreases the rates of protein-catalyzed phospholipid transfer. The potency of inhibition is dibucaine>tetracaine>lidocaine>procaine, an order which is compared with and identical to those for a wide variety of anesthetic-dependent membrane phenomena. Half-maximal inhibition of phosphatidylinositol transfer by dibucaine occurs at a concentration of 0.18 mM, significantly lower than the concentration of 1.9 mM required for half-maximal inhibition of phosphatidylcholine transfer activity of the brain protein. Comparable inhibition of liver protein phosphatidylcholine transfer activity is observed at 1.6 mM dibucaine. For activity measurements performed at different pH, dibucaine is more potent at the lower pH values which favor the equilibrium toward the charged molecular species. With membranes containing increasing molar proportions of phosphatidate, dibucaine is increasingly more potent. No effect of Ca²⁺ on the control transfer activity or the inhibitory action of dibucaine is noted. These results are discussed in terms of the formation of specific phosphatidylinositol or phosphatidylcholine complexes with the amphiphilic anesthetics in the membrane bilayer.     

Studies on Peptidases of Aspergillus oryzae

The homogeneity of Aspergillus dipeptidase prepared according to the standard method established by us was ascertained by ultracentrifugation and some characteristic properties of the enzyme was further investigated.

Hydrolysis of various dipeptides by the purified dipetidase was tested in the presence of divalent metal ions such as Co⁺⁺ or Zn⁺⁺, and the characteristics of greatest interest may be enumerated as follows:

1. The homogeneous dipeptidase requires Zn⁺⁺ for activation in the case of the hydrolysis of leucylglycine, leucylalanine leucylleucine, etc.

2. The homogeneous dipeptidase requires Co⁺⁺ for activation in the case of the hydrolysis of glycylleucine, glycylleucine, glycylglycine, glycylphenylalanine, etc.

3. In the case of the hydrolysis of alanylglycine, alanylleucine, valylglycine, etc., this enzyme does not require any metal ions.

     

Studies on Peptidases of Aspergillus oryzae

Screening experiments for dipeptidase and aminopolypeptidase from 40 strains of molds were conducted using Leu-Gly, Gly-Leu, Ala-Gly and Gly-Gly-Leu as substrates.

The strains of Aspergillus oryzae RO-0129 A-2, IAM-2600 and IAM-2616 showed strong activities of both dipeptidase and aminopolypeptidase.

Further, optimal conditions for making culture as well as those for the extractions of the peptidases from the mycerial mats were investigated.     

Plasma cholesteryl ester transfer protein mass and phospholipid transfer protein activity are associated with leptin in type 2 diabetes mellitus

Adipose tissue contributes to plasma levels of lipid transfer proteins and is also the major source of plasma adipokines. We hypothesized that plasma cholesteryl ester transfer protein (CETP) mass, phospholipid transfer protein (PLTP) activity and cholesteryl ester transfer (CET, a measure of CETP action) are determined by adipokine levels. In this study, relationships of plasma CETP mass, PLTP activity and CET with leptin, resistin and adiponectin were analyzed in type 2 diabetic patients and control subjects. Plasma PLTP activity (P<0.001), CET (P<0.001), leptin (P=0.003), resistin (P<0.001), high sensitive C-reactive protein (P=0.005), and insulin resistance (HOMA(ir)) (P<0.001) were higher, whereas HDL cholesterol (P<0.001) and plasma adiponectin (P<0.001) were lower in 83 type 2 diabetic patients (32 females) than in 83 sex-matched control subjects. Multiple linear regression analysis demonstrated that in diabetic patients plasma leptin levels were related to plasma CETP mass (P=0.018) and PLTP activity (P<0.001), but not to the other adipokines measured. Plasma CET was inversely correlated with adiponectin in univariate analysis, but this association disappeared in multivariate models that included plasma lipids and CETP. In conclusion, both plasma CETP mass and PLTP activity are associated with plasma leptin in type 2 diabetes. The elevated CET in these patients is not independently related to any of the measured plasma adipokines.     

Isolation and characterization of Aspergillus oryzae vacuolar protein sorting mutants

The vacuolar protein sorting (vps) system in the filamentous fungus Aspergillus oryzae, which has unique cell polarity and the ability to secrete large amounts of proteins, was evaluated by using mutants that missort vacuolar proteins into the medium. Vacuolar carboxypeptidase Y (CPY) fused with enhanced green fluorescent protein (EGFP) was used as a vacuolar marker. Twenty dfc (dim EGFP fluorescence in conidia) mutants with reduced intracellular EGFP fluorescence in conidia were isolated by fluorescence-activated cell sorting from approximately 20,000 UV-treated conidia. Similarly, 22 hfm (hyper-EGFP fluorescence released into the medium) mutants with increased extracellular EGFP fluorescence were isolated by using a fluorescence microplate reader from approximately 20,000 UV-treated conidia. The dfc and hfm mutant phenotypes were pH dependent, and missorting of CPY-EGFP could vary by 10- to 40-fold depending on the ambient pH. At pH 5.5, the dfc-14 and hfm-4 mutants had an abnormal hyphal morphology that is consistent with fragmentation of vacuoles and defects in cell polarity. In contrast, the hyphal and vacuolar morphology of the dfc-14 and hfm-4 mutants was normal at pH 8.0, although CPY-EGFP accumulated in perivacuolar dot-like structures similar to the class E compartments in Saccharomyces cerevisiae vps mutants. In hfm-21, CPY-EGFP localized at the Spitzenk?rper when the mutant was grown at pH 8.0 but not in vacuoles, suggesting that hfm-21 may transport CPY-EGFP via a novel pathway that involves the Spitzenk?rper. Correlations between vacuolar protein sorting, pH response, and cell polarity are reported for the first time for filamentous fungi.     

Different phospholipid transfer protein complexes contribute to the variation in plasma PLTP specific activity

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids among lipoproteins. Over half of the PLTP in human plasma has been found to have little phospholipid transfer activity (inactive PLTP). We recently observed that plasma PLTP specific activity is inversely correlated with high-density lipoprotein (HDL) level and particle size in healthy adults. The purpose of this study was to evaluate the factors that contribute to the variation in plasma PLTP specific activity. Analysis of the specific activity of PLTP complexes in nine plasma samples from healthy adults revealed two clusters of inactive PLTP complexes with mean molecular weights (MW) of 342kDa and 146kDa. The large and small inactive PLTP complexes represented 52±8% (range 39-63%) and 8±8% (range 1-28%) of the plasma PLTP, respectively. Active PLTP complexes had a mean MW of 207kDa and constituted 40±6% (range 33-50%) of the plasma PLTP. The specific activity of active PLTP varied from 16 to 32μmol/μg/h. These data demonstrate for the first time the existence of small inactive plasma PLTP complexes. Variation in the amount of the two clusters of inactive PLTP complexes and the specific activity of the active PLTP contribute to the variation in plasma PLTP specific activity.     

Stable high-copy-number integration of Aspergillus oryzae alpha-AMYLASE cDNA in an industrial baker's yeast strain.

The Aspergillus oryzae alpha-amylase cDNA was placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into the ribosomal DNA locus of an industrial baker's yeast strain. To obtain a strain eligible for commercial use, we constructed an integrative cassette lacking bacterial DNA sequences but containing the alpha-amylase cDNA and ribosomal DNA sequences to target the integration to this locus. High-copy-number integrants were obtained including a defective TRP1d promoter in the integrative cassette. We selected one transformant, Rib-AMY (CECT10872), in which the multi-integrated sequences were stable even after 200 generations of growth in nonselective medium. This transformant also expressed and secreted high levels of alpha-amylase. Bread made with this strain had a higher volume, lower density, and softer crumbs than bread made with a control strain. The Rib-AMY transformant also was useful in retarding bread firming. This new strain fulfills all the requirements for commercial utilization and should reduce or eliminate the requirement for addition of exogenous alpha-amylase to the flour, reducing allergenic work-related symptoms due to this enzyme.