Soybean (Glycine max (L.) Merr) is the largest oil and protein crop in the world and it is grown for both oil and protein. To address the needs of both the edible oil market and industrial applications of soybean oil, fatty acid modification has been a focus of soybean breeding programs. Natural variation, mutagenesis and genetic engineering have been used to alter the fatty acid profile. Several genes, mostly desaturases, have been associated with altered fatty acid profiles but enzymes in the Kennedy Pathway have yet to be studied as another source of genetic variation for altering the fatty acid profiles. The Kennedy Pathway is also known as the oil producing pathway and consists of four enzymes: glycerol-3-phosphate acyltransferase (G3PAT); lysophosphatidic acid acyltransferase (LPAAT); phosphatidic acid phosphatase (PAP); and diacylglycerol acyltransferase 1 (DGAT1). The starting material for this pathway is glycerol-3-phosphate, which is produced from glycerol by glycerol kinase (GK), and the product of this pathway is triacylglycerol (TAG). The overall objective of this study was to elucidate the role that the Kennedy Pathway plays in determining the fatty acid profile in two ways: (1) sequencing the transcribed region of the genomic genes encoding the enzymes of GK, G3PAT, LPAAT, and DGAT1 in soybean genotypes with altered fatty acid profiles; and (2) studying their expression over seed development, across three growing temperatures. The genetic material for the study consisted of four soybean genotypes with altered fatty acid profile: RG2, RG7, RG10, and SV64-53. Results from sequencing showed that the mutations identified in G3PAT, LPAAT, and DGAT1 in the four soybean genotypes did not explain the differences in the fatty acid profiles. The expression of G3PAT, LPAAT, and DGAT1 over seed development showed that G3PAT had the lowest levels, followed by LPAAT, then DGAT1, across the growing temperatures. The differences in expression among genotypes corresponded to differences in fatty acid accumulation, suggesting that expression rather than genetic mutations in the transcribed region of the genes influenced the fatty acid profile of the genotypes in this study. In conclusion, the enzymes of the Kennedy Pathway appear to contribute to the altered fatty acid profiles observed in the soybean mutant genotypes. / Ontario Ministry of Economic Development and Innovation (formerly Ontario Ministry of Research and Innovation), BioCar Initiative, Grain Farmers of Ontario, SeCan

Lecithin:cholesterol acyltransferase (LCAT) was isolated from hog plasma and basic physicochemical properties and functionally important regions were investigated. Approximately one milligram of the enzyme was purified to apparent homogeneity with approximately a 20,000-fold increase in specific activity. In the plasma, hog LCAT was found to associate with high-density lipoproteins (HDL) probably through hydrophobic interactions with apolipoprotein A-I. HDL was the preferred lipoprotein substrate of the enzyme as its macromolecular substrate. The enzyme was found to contain 4 free sulfhydryl groups; at least one of these appeared to be essential for catalytic activity. The enzyme had a tendency to aggregate at high concentrations. More than half of the tryptophan and none of the tyrosine residues of the enzyme were shown to be exposed to the aqueous environment based on fluorescence and absorbance studies, respectively.

Hemolysin toxin produced and secreted by pathogenic Escherichia coli is one of a family of cytolytic, structurally homologous protein toxins known as RTX (repeats in toxin) toxins. RTX toxins are products of a gene cluster, CABD . The A gene product, nontoxic hemolysin (proHlyA) is made toxic by post-translational fatty acylation of two internal lysine residues. HlyC, C gene product, is essential for acylation, and acyl-acyl carrier protein (ACP) is the acyl donor. HlyB and HlyD are involved in secretion of the toxin. HlyC was thought to serve as an internal protein acyltransferase and remained uncharacterized until now. ProHlyA and HlyC were separately subcloned, expressed, and purified, and acyl-ACPs with diverse radioactive acyl groups were synthesized. With these proteins, the conversion of proHlyA to HlyA by acyltransfer was assayed. Acyl-ACP was the obligate acyl donor. Acyltransfer was catalyzed by HlyC monomer, and an acyl-enzyme intermediate was detected and shown to catalyze the reverse reaction. The reaction mechanism was examined by steady state kinetics, and the nature of inhibitions by reaction products was determined. The kinetic mechanism of the internal protein acylation was compatible with an uni uni iso uni uni ping pong with isomerization of the F form of the enzyme. Clues to the chemical mechanism for the acyltransferase were elucidated by both chemical modification studies and site directed mutagenesis of the enzyme. Chemical modification experiments ruled out any critical cysteines, serines, and lysine residues, but suggested a role for histidine(s) and tyrosine(s) in acyltransferase function. In order to examine the function of specific residues and possibly corroborate the chemical findings, site directed mutagenesis studies of the acyltransferase were employed. Seventeen residues that were conserved among 13 different RTX toxin acyltransferases were individually mutated, and the respective HlyCs expressed, and characterized. Residues that were critical for acyltransferase function included Gly 11, His 23, Tyr 70, and Gly 85. As with chemical modification data, mutagenesis ruled out any conserved, essential, cysteines or serines critical for HlyC acyltransferase activity.

Triacylglycerols are the predominant molecules of energy storage in eukaryotes. Triacylglycerol synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Although the use of molecular tools, including targeted disruption of either DGAT enzyme, has shed light on their metabolic functions, little is known about the mechanisms responsible for regulating DGAT activity. Several lines of evidence from previous studies have suggested that DGAT1, but not DGAT2, is subject to regulation by phosphorylation and that protein kinase A (PKA)-dependent pathways are likely involved. In this study, the role of PKA in regulating DGAT activity and triacylglycerol synthesis during lipolysis was investigated. By using 3T3-L1 adipocytes, in vitro DGAT activity was shown to increase 2 fold during lipolysis. This data suggests that PKA might phosphorylate and activate DGAT1 during lipolysis to promote the recycling/re-esterification of excessive free fatty acids into triacylglycerols before they reach toxic levels within the cell. Additionally, high-performance liquid chromatography electrospray ionization mass spectrometry/mass spectrometry was exploited to identify PKA phosphorylation sites of DGAT1, and serine-17, -20 and -25 were identified as potential PKA phosphorylation sites using this methodology. The functional importance of these three potential phosphorylation sites was examined. Mutations of these sites to alanines (to prevent phosphorylation) or aspartates (to mimic phosphorylation) gave rise to enzymes functioning similarly to wild-type DGAT1. These phosphorylation sites appeared to be functionally silent as they were not involved in regulating DGAT1 activity, multimer formation, or enzyme stability. However, PKA phosphorylation at these three sites seemed to play a role in affinity of DGAT1 for its diacylglycerol substrate. These results indicate the existence of other unidentified, functionally active PKA phosphorylation sites or phosphorylation sites of other kinases, which are involved in regulating DGAT1.

Triacylglycerols are the predominant molecules of energy storage in eukaryotes. Triacylglycerol synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Although the use of molecular tools, including targeted disruption of either DGAT enzyme, has shed light on their metabolic functions, little is known about the mechanisms responsible for regulating DGAT activity. Several lines of evidence from previous studies have suggested that DGAT1, but not DGAT2, is subject to regulation by phosphorylation and that protein kinase A (PKA)-dependent pathways are likely involved. In this study, the role of PKA in regulating DGAT activity and triacylglycerol synthesis during lipolysis was investigated. By using 3T3-L1 adipocytes, in vitro DGAT activity was shown to increase 2 fold during lipolysis. This data suggests that PKA might phosphorylate and activate DGAT1 during lipolysis to promote the recycling/re-esterification of excessive free fatty acids into triacylglycerols before they reach toxic levels within the cell. Additionally, high-performance liquid chromatography electrospray ionization mass spectrometry/mass spectrometry was exploited to identify PKA phosphorylation sites of DGAT1, and serine-17, -20 and -25 were identified as potential PKA phosphorylation sites using this methodology. The functional importance of these three potential phosphorylation sites was examined. Mutations of these sites to alanines (to prevent phosphorylation) or aspartates (to mimic phosphorylation) gave rise to enzymes functioning similarly to wild-type DGAT1. These phosphorylation sites appeared to be functionally silent as they were not involved in regulating DGAT1 activity, multimer formation, or enzyme stability. However, PKA phosphorylation at these three sites seemed to play a role in affinity of DGAT1 for its diacylglycerol substrate. These results indicate the existence of other unidentified, functionally active PKA phosphorylation sites or phosphorylation sites of other kinases, which are involved in regulating DGAT1.

Vitamin C (ascorbate and dehydroascorbic acid) is vital for plants and found throughout the plant cell including in the apoplast. The structure of ascorbate was determined eighty years ago; however, many of its degradation pathways remain unclear. Numerous degradation products of ascorbate have been reported to occur in the apoplast but many still remained unidentified. Ascorbate is well known as an antioxidant, and acts to quench reactive oxygen species (ROS), such as hydrogen peroxide and ozone in the plant apoplast. The immediate oxidation product of ascorbate is dehydroascorbic acid (DHA), which may be quickly hydrolysed to diketogulonic acid (DKG). The further reactions of radiolabelled and non-radiolabelled DHA and DKG with various ROS have been investigated. Differences were observed in the products formed from the various ROS, allowing a unique fingerprint of oxidation products to be described for each ROS. Equally, different compounds were produced depending on the starting substrate; for example cyclic oxalyl threonate was only observed in the reactions of DHA and not DKG. A major oxidation product of DHA is OxT. A novel enzyme activity involving the transfer of the oxalyl group from OxT to an acceptor substrate such as a sugar has been detected. This enzyme activity could have potential cell wall modification roles, in the formation of oxalate cross-linkages between cell wall components. This would provide a novel role for ascorbate derivatives in cell growth. Vitamin C is also a vital component of the human diet, and most dietary ascorbate comes from plants such as salads. The degradation of ascorbate during post-harvest processing and storage of salad leaves has been investigated. Spinach leaves were found to be particularly prone to losing ascorbate during the industrial washing process. The use of radiolabelled ascorbate has allowed the determination that the major degradation product formed from ascorbate during spinach washing was oxalate.

Cytosolic LPA acyltransferase in yeast An isooctane tolerant strain of S. cerevisiae KK-12 was reported to have increased saturated fatty acid content (Miura et. al., 2000). Amongst the various genes upregulated on isooctane treatment, ICT1 (Increased Copper Tolerance 1) was found to have maximal expression (Miura et. al., 2000; Matsui et. al., 2006). This gene in S. cerevisiae is encoded by YLR099C annotated as Ict1p. However, the physiological significance of Ict1p was not understood. Here we showed that an increase in the synthesis of phosphatidic acid (PA) is responsible for enhanced phospholipid synthesis, which confers organic solvent tolerance to S. cerevisiae. This increase in the PA formation is due to the upregulation of Ict1p, a soluble oleoyl-CoA dependent lysophosphatidic acid (LPA) specific acyltransferase. Analysis of Δict1 strain by in vivo [32P]orthophosphate labeling showed a drastic reduction in PA, suggesting the role of Ict1p in phospholipid biosynthesis. Overexpression of Ict1p in S. cerevisiae showed an increase in PA and the overall phospholipid content on organic solvent exposure. The purified recombinant enzyme was found to specifically acylate LPA. Specific activity of Ict1p was found to be higher for oleoyl-CoA as compared to palmitoyl-CoA and stearoyl-CoA. The study therefore, provides a mechanistic basis of solvent tolerance in S. cerevisiae.It is well known that phosphatidic acid (PA) is formed by the acylation of LPA by LPA acyltransferase. However, all the LPA acyltransferases characterized till date have distinct transmembrane domains and form a member of membrane bound biosynthetic machinery of phospholipid biosynthesis. They have a conserved signature motif, H(X)4D. Phosphatidic acid is an important precursor for the synthesis of glycerophospholipids and triacylglycerols. PA enters the biosynthetic pathway of phospholipids through a CTP-dependent activation catalyzed by CDPdiacylglycerol synthase. This enzyme forms CDP-diacylglycerol, which serves as a direct precursor for phosphatidylinositol, phosphatidylglycerol and cardiolipin. PA can also be dephosphorylated by phosphatidic acid phosphatase yielding diacylglycerol, which serves as a precursor for the formation of PE and PC through the CDP-ethanolamine and CDP-choline pathway or for the triacylglycerol synthesis through a dephosphorylation step followed by an acylation establishing it as a supreme molecule for the acylglycerol biosynthesis. Since, PA is an important intermediate and that there are mechanisms to synthesize PA, other than the conventional membrane bound pathways, we wanted to understand whether such a mechanism of PA biosynthesis is conserved across the plant and animal kingdom. Therefore, we resorted to analyze Ict1p like proteins in Arabidopsis and human whose complete genome sequence is available. Cytosolic LPA acyltransferase in Arabidopsis Homology search with ICT1 in Arabidopsis thaliana genome, led to the identification of At4g24160 as a close relative. In order to gain an insight into the significance of such proteins in plants we performed a genome wide survey of At4g24160 like proteins in Arabidopsis. We identified that A. thaliana genome encodes twenty four At4g24160 like proteins, most of which belong to the α/β- hydrolase family of proteins and possess a distinct lipase motif (GXS/NXG). Interestingly, amongst these twenty four, only At4g24160 has a conserved HX4D motif. Domain analysis of these proteins suggests a wide functional diversification during evolution. Gene expression studies revealed their importance during various abiotic stress. Bacterial expression of At4g24160 followed by its purification using Ni2+-NTA column chromatography and characterization revealed it to be a LPA acyltransferase. Expression analysis showed that it is highly expressed in the pollen grains followed by the root cap. In addition, the gene was found to be upregulated under salt stress conditions. Direct correlation between salt stress and phospholipid biosynthesis is well known in the literature. We envisage that At4g24160 might be one of the gene products involved in membrane repair when exposed to such a stressCytosolic LPA acyltransferase in human Homology search with Ict1p revealed another interesting candidate protein in Homo sapiens known as Comparative Gene Identification–58 (cgi-58). Mutations in CGI- 58 are known to be the causative reason for a rare autosomal recessive genetic disorder known as Chanarin-Dorfman syndrome characterized by the excessive TG accumulation and defective membrane phospholipid regulation in several tissues. It is known to be a coactivator of adipose triglyceride lipase (ATGL), promoting lipolysis of TG (Lass et. al., 2006). However, the exact biochemical role remains unknown. To understand the biochemical function of cgi-58, the gene was overexpressed in E. coli and the purified, recombinant protein was found to specifically acylate lysophosphatidic acid in an acyl-CoA dependent manner. Overexpression of CGI-58 in Δict1 rescued the metabolic defect of the strain. Heterologous overexpression of CGI-58 in S. cerevisiae followed by metabolic labeling with [32P]orthophosphate showed an increased biosynthesis of membrane phospholipids. Analysis of neutral lipid biosynthesis by [14C]acetate labeling showed an increase in DG and free fatty acids. However, marked decrease in the TG biosynthesis was seen. Decrease in TG was confirmed by ESI-MS. In addition, physiological significance of cgi-58 in the mice white adipose tissue is reported in this thesis. We found soluble lysophosphatidic acid acyltransferase activity in the mice white adipose tissue. Immunoblot with anti-Ict1p antibodies followed by MALDI-TOF analysis of the cross reacting protein in lipid droplets revealed its identity as cgi-58. These observations suggest the existence of an alternate cytosolic phosphatidic acid biosynthetic pathway in the white adipose tissue. Collectively, our observations suggest a possible involvement of cgi-58 in the phospholipid biosynthesis of adipocytes and its probable role in maintaining the TG homeostasis. In conclusion, the study reveals the significance of cytosolic lipid metabolic enzymes having conserved biochemical function, in maintaining homeostasis in living organisms across phylogeny.

The skin of mammals contains sebaceous glands (SGs) which are attached to the hair follicle (HF), and their best known function is to release sebum onto the skin surface via the HF canal. It has long been known that these two entities of the pilosebaceous unit are interconnected, but the extent to which the two ‘control’ one another was less clear. The current project set out to investigate the impact of the HF cycle on the SG. It was found that in a depilation- induced HF cycle, SG morphology altered drastically, with an increase in SG area (P<0.001), number of sebocytes (P<0.001), and individual sebocyte area (P<0.001) occurring after HF depilation. In SGs attached to a spontaneously cycling HF, none of the above was observed, indicating that spontaneous HF cycling does not affect SG morphology, whereas anagen induction by depilation is associated with altered SG morphology, likely as a result of HF trauma. Diacylglycerol acyltransferase 1 (DGAT1) is an enzyme known for its role in the production of various lipids. It was previously shown that DGAT1 knockout in mice caused SG atrophy, which was thought to be caused by an increased level of retinoic acid within the skin, which in turn caused atrophy of the gland. The current project aimed to further investigate the role of the DGAT1 enzyme in murine skin. Based on the results of the previous experiments, HF and SG morphology of spontaneously-cycling DGAT1 knockout mice were assessed. It was found that DGAT1 knockout caused delayed HF morphogenesis, altered HF cycling, increased HF length (P<0.001), more acute HF growth angle (P<0.001), increased SG apoptosis(P<0.001), decreased SG lipid content (P<0.001) and dysfunctional lipid droplet formation. The impact of DGAT1 knockout on HF morphology and cycling suggests that DGAT1 knockout causes alterations in the WNT/ beta-catenin signalling pathway, as these processes are highly controlled by this signalling pathway. In order to investigate the role of the DGAT1 enzyme in human HFs, and to investigate the hypothesis that DGAT1 may directly interact with the WNT/ beta-catenin signalling pathway, HFs were organ-cultured in the presence of a pharmacological DGAT1 inhibitor (AZD7687). It was found, at the transcriptional level, that one of the major canonical pathways affected by DGAT1 inhibition in human HFs was the WNT/ beta-catenin signalling pathway. DGAT1 inhibition was found to cause suppression of the WNT/beta-catenin signalling pathway via a down-regulation of a number of WNT/beta-catenin related genes. Overall, these results show that SG morphology is largely dependent upon HF homeostasis, and suggest that the DGAT1 enzyme may possess a previously unknown role, directly impacting the WNT/ beta-catenin signalling pathway.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21841号 / 農博第2354号 / 新制||農||1069(附属図書館) / 学位論文||H31||N5213(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 植田 充美, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The avocado mesocarp contains up to 60-70% oil by dry weight where triacylglycerol (TAG) is the major constituent. This neutral lipid, TAG is utilized by plants for the carbon and energy source when stores in seed tissue. There is significant human nutritional demand for vegetable oil, but its use in production of renewable biomaterials and fuels has intensified the need to increase oil production. In plants, the final and committed step in TAG biosynthesis is catalyzed by diacylglycerol acyltransferases (DGAT) and/or a phospholipid: diacylglycerol acyltransferases (PDAT). Both DGAT and PDAT contribute to seed TAG biosynthesis in an independent or overlapping manner, depending on the species. However, the regulation of TAG biosynthesis is not wellstudied in nonseed tissues such as mesocarp of avocado. Based on the transcriptome data of Persea americana it is hypothesized that both DGAT and PDAT are likely to catalyze the conversion of diacylglycerol to TAG. In this study, putative DGAT1 was identified and comprehensive in silico analyses were conducted to determine the respective start codons, full-length coding sequences, transmembrane domains, predicted protein structures and phylogenetic relationships with other known DGAT1s. These data reveal that the putative DGATs of a basal angiosperm species retain features that are conserved not only among angiosperms but also other eukaryotes. For further functional analysis, the avocado DGAT1 was expressed in H1246, a TAG-deficient yeast strain and lipotoxicity rescue assays, TLC analysis, Nile Red staining were conducted. The complementation of this yeast strain confirmed enzyme activity and supported the possible role of avocado DGAT1 in TAG biosynthesis. Finally, substrate specificity of DGAT was determined by incubating microsomes with different radiolabeled substances and found that avocado DGAT1 has a preference toward oleic acid (18:1) compare to palmitic acid (16:0) while it is converting diacylglycerol (DAG) to triacylglycerol. All these data suggested that avocado DGAT1 is functional and making TAG with high preference of oleic acid over palmitic acid.