Man-Jong Kang

Molecular Characterization and Expression of Rat Acyl-CoA Synthetase 3

Isolation and characterization of a rat brain cDNA identified a third acyl-CoA synthetase (ACS) designated ACS3. The deduced amino acid sequence of the cDNA revealed that ACS3 consists of 720 amino acids and exhibits a structural architecture common to ACSs from various origins. ACS3 expressed in COS cells was purified to near homogeneity. The purified ACS3 resolved by SDS-polyacrylamide gel electrophoresis into two major proteins of 79 and 80 kDa. Cell-free translation of a synthetic mRNA encoding the entire region of ACS3 revealed that the two isoforms were derived from the same mRNA. The purified ACS3 utilizes laurate and myristate most efficiently among C8-C22 saturated fatty acids and arachidonate and eicosapentaenoate among C16-C20 unsaturated fatty acids. Northern blot analysis revealed that ACS3 mRNA is most abundant in brain and, to a much lesser extent, in lung, adrenal gland, kidney, and small intestine. During the development of the rat brain, expression of ACS3 mRNA reached a maximum level at 15 days after birth and then declined gradually to 10% of the maximum in the adult brain.

Expression Cloning and Characterization of a Novel Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein, GPI-HBP1

By expression cloning using fluorescent-labeled high density lipoprotein (HDL), we isolated two clones that conferred the cell surface binding of HDL. Nucleotide sequence of the two clones revealed that one corresponds to scavenger receptor class B, type 1 (SRBI) and the other encoded a novel protein with 228 amino acids. The primary structure of the newly identified HDL-binding protein resembles GPI-anchored proteins consisting of an N-terminal signal sequence, an acidic region with a cluster of aspartate and glutamate residues, an Ly-6 motif highly conserved among the lymphocyte antigen family, and a C-terminal hydrophobic region. This newly identified HDL-binding protein designated GPI-anchored HDL-binding protein 1 (GPI-HBP1), was susceptible to phosphatidylinositol-specific phospholipase C treatment and binds HDL with high affinity (calculated K d = 2–3 μg/ml). Similar to SRBI, GPI-HBP1 mediates selective lipid uptake but not the protein component of HDL. Among various ligands for SRBI, HDL was most preferentially bound to GPI-HBP1. In contrast to SRBI, GPI-HBP1 lacked HDL-dependent cholesterol efflux. The GPI-HBP1 transcripts were detected with the highest levels in heart and, to a much lesser extent, in lung and liver. In situhybridization revealed the accumulation of GPI-HBP1 transcripts in cardiac muscle cells, hepatic Kupffer cells and sinusoidal endothelium, and bronchial epithelium and alveolar macrophages in the lung.

Fatty acid induced glioma cell growth is mediated by the acyl-CoA synthetase 5 gene located on chromosome 10q25.1-q25.2, a region frequently deleted in malignant gliomas

Acyl-CoA synthetase (ACS) ligates fatty acid and CoA to produce acyl-CoA, an essential molecule in fatty acid metabolism and cell proliferation. ACS5 is a recently characterized ACS isozyme highly expressed in proliferating 3T3-L1 cells. Molecular characterization of the human ACS5 gene revealed that the gene is located on chromosome 10q25.1-q25.2, spans approximately 46 kb, comprises 21 exons and 22 introns, and encodes a 683 amino acid protein. Two major ACS5 transcripts of 2.5- and 3.7-kb are distributed in a wide range of tissues with the highest expression in uterus and spleen. Markedly increased levels of ACS5 transcripts were detected in a glioma line, A172 cells, and primary gliomas of grade IV malignancy, while ACS5 expression was found to be low in normal brain. Immunohistochemical analysis also revealed strong immunostaining with an anti-ACS5 antibody in glioblastomas. U87MG glioma cells infected with an adenovirus encoding ACS5 displayed induced cell growth on exposure to palmitate. Consistent with the induction of cell growth, the virus infected cells displayed induced uptake of palmitate. These results demonstrate a novel fatty acid-induced glioma cell growth mediated by ACS5.

Roles of Apolipoprotein E Receptors in Lipoprotein Metabolism

Two apolipoprotein E (apoE) specific receptors, designated as ‘very low density lipoprotein receptor’ (VLDLR) and ‘apoE receptor 2’ (apoER2), closely resemble the structure of the low density lipoprotein receptor (LDLR), but they are expressed in different tissues. VLDLR mRNA is abundant in heart, skeletal muscle, brain and adipose tissues, whereas apoER2 mRNA predominates in the brain. In chicken, VLDLR is expressed almost exclusively in oocytes and mediates the uptake of yolk precursors, VLDL and vitellogenin. In contrast to the chicken, where absence of VLDLR results in an inability to lay eggs, the phenotype of VLDLR knockout mice is rather modest, except for a marked reduction of triacylglycerol content in adipose tissue, suggesting that VLDLR plays a part in the uptake of fat into adipocytes. Characterization of the apoER2 gene in various species has revealed the occurrence of an exon loss during its evolution and suggests that exon loss has contributed to the evolution of genes containing repetitive sequences. In addition to apoER2 and VLDLR, we have identified a new LDLR related protein (LRP) that recognizes apoE. This new LRP is expressed in the liver and steroidogenic tissues, suggesting a role in the clearance of apoE-lipoproteins.

Molecular Characterization of Arachidonate Preferring Acyl-CoA Synthetase, ACS4

In arachidonate metabolism, acyl-CoA synthetase(ACS) plays a key role in esterification of free arachidonate into membrane phospholipids. ACS4 is an arachidonate preferring ACS abundant in steroidogenic tissues. The human gene for ACS4 (FACL4) is mapped chromosome band Xq22-23, a region close to the a5 chain of the type IV collagen gene that is related to X-linked Alport Syndrome. Recent studies have revealed that FACL4 is deleted in patients with Alport Syndrome, elliptocytosis and mental retardation, suggesting that the enzyme may play a role in intellectual growth in humans.