An alternatively processed mRNA specific for gamma-glutamyl transpeptidase in human tissues

Human gamma-glutamyl transpeptidase (GGT)1 is composed of two subunits derived from a single precursor (Nash, B., and Tate, S.S. (1984) J. Biol. Chem. 259, 678-685; Finidori, J., Laperche, Y., Tsapis, R., Barouki, R., Guella?n, G., and Hanoune, J. (1984) J. Biol. Chem. 259, 4687-4690) consisting of 569 amino acids (Laperche, Y., Bulle, F., Aissani, T., Chobert, M.N., Aggerbeck, M., Hanoune, J., and Guella?n, G. (1986) Proc Natl. Acad. Sci. U.S.A. 83, 937-941). In the present study we report the cloning of an altered form of this precursor from human liver. We have isolated two clones, one 2,632 base pairs (bp) long from a fetal liver cDNA library and one 926 bp long from an adult liver cDNA library, each containing a 22-bp insertion that introduces a premature stop codon and shortens the open reading frame to 1,098 bp when compared with known human cDNA sequences specific for GGT. Sequence analysis of a human genomic GGT clone shows that this insertion of 22 bp is generated by a splicing event involving an alternative 3'-acceptor site. By polymerase chain reaction experiments we demonstrate that the alternatively spliced mRNA is present in polysomes from the microsomal fraction of a human hepatoma cell line (Hep G2) and thus could encode an altered GGT molecule of 39,300 Da (366 amino acids) encompassing most of the heavy subunit which is normally 41,500 Da (380 amino acids). The altered mRNA is detected in various human tissues including liver, kidney, brain, intestine, stomach, placenta, and mammary gland. This report is the first demonstration of an alternative primary sequence in the mRNA coding for GGT, a finding that could be related to the presence of some inactive forms of GGT detected in human tissues.     

人细胞内胆固醇酯化酶ACAT

酰基辅酶A:胆固醇酰基转移酶(ACAT)是细胞内唯一催化游离胆固醇与长链脂肪酸形成胆固醇酯的酶,在维持细胞内胆固醇、脂肪酸等脂质代谢平衡中起着极重要的作用。人类一些重要疾病如心脑血管病的动脉粥样硬化、神经系统的阿尔茨海默症、消化吸收系统的胆囊病变等都与ACAT紧密关联。本文主要从人ACAT的表达、功能效应、抑制剂应用前景等三方面介绍有关的研究进展。     

Expression levels of ACAT1 and ACAT2 genes in the liver and intestine of baboons with high and low lipemic responses to dietary lipids

Acyl–coenzyme A:cholesterol acyltransferase (ACAT) 1 and ACAT2 play an important role in cellular cholesterol esterification and thus modulate intestinal cholesterol absorption and hepatic lipoprotein secretion. The relative expression levels of ACAT1 and ACAT2 in human tissues differ from those in other animals, including nonhuman primates. The present study compared the relative expression levels of ACAT1 and ACAT2 in baboons with high and low lipemic responses to dietary lipids. We isolated RNA and prepared cDNA from frozen liver and small intestine from high- and low-responding pedigreed baboons necropsied after consuming a high-cholesterol and high-fat diet for 18 months. The expression of ACAT1 and ACAT2 was measured by TaqMan real-time quantitative PCR normalized to 18s ribosomal RNA. The expression of ACAT1 was higher than that of ACAT2 in the liver, whereas the expression of ACAT2 was higher than that of ACAT1 in the duodenum and jejunum. There was no difference in the expression of ACAT1 or ACAT2 in the liver and intestine between high- and low-responding baboons except that the expression of ACAT1 was higher in the duodenum of high responders than in that of low responders. Western blot analysis also showed a higher level of ACAT1 protein in the duodenum of high responders than in that of low responders. There was a significant correlation between duodenal ACAT expression levels and total plasma cholesterol concentration in baboons. These results suggest that differences in ACAT1 expression may affect plasma cholesterol concentration and partly affect diet-induced hyperlipidemia.     

Acyl-CoA: cholesterol acyltransferase-2 gene polymorphisms and their association with plasma lipids and coronary artery disease risks

Acyl-CoA: cholesterol acyltransferase-2 (ACAT2), an intracellular cholesterol esterification enzyme found only in the intestine and liver, has been demonstrated to be associated with hypercholesterolemia and atherosclerosis in mice. To explore the possible impact of ACAT2 gene variants on CAD susceptibility and plasma lipid levels, three polymorphisms, 41A>G (Glu>Gly), 734C>T (Thr>Ile), and IVS4-57_58 ins48 bp (D/I), were genotyped in 809 CAD patients (CAD+) and 1,304 controls (CAD−) from three distinct Singaporean ethnic groups (1,228 Chinese, 367 Malays and 518 Indians). The 734T allele frequency was significantly lower in CAD+ (0.20) than CAD− (0.26) in Chinese (P=0.003) and I allele of D/I was significantly higher in CAD+ (0.17) than CAD− (0.10) in Indians (P=0.011). The 41G allele was significantly more frequent among normolipidemic (0.19) than dyslipidemic (0.13) individuals in Chinese (P=0.008). In normolipidemic females, 734C>T was associated with apoA1, apoB and lipoprotein (a) in Indians, and with apoA1 in Malays, whereas 41A>G is associated with total cholesterol in Indians. The 734C>T polymorphism was in almost complete linkage disequilibrium (LD) with the IVS4-57_58 ins48 bp and in very strong LD with 41A>G in all the three ethnic groups. In the normolipidemic females, the AG/CT had much higher apoB than AA/CC in Indians. We found that the three ACAT2 polymorphisms studied are associated with CAD risk and plasma lipid levels but their effects are not consistent across genders and ethnic groups.     

Two newly identified exons in human GRM1 express a novel splice variant of metabotropic glutamate 1 receptor

To date, five human metabotropic glutamate (mGlu) 1 receptor splice variants (1a, 1b, 1d, 1f, and 1g) have been described, all of which involve alternative C-terminal splicing. mGlu1a receptor contains a long C-terminal domain (341 amino acids), which has been shown to scaffold with several proteins and contribute to the structure of the post-synaptic density. However, several shorter mGlu1 receptor splice variants lack the sequence required for these interactions, and no major functional differences between these short splice variants have been described. By using RT-PCR we have shown that two human melanoma cell lines express both mGlu1a and mGlu1b receptors. In addition, using 3′RACE, we identified three previously unknown mGlu1 receptor mRNAs. Two differ in the length of their 3′ untranslated region (UTR), and encode the same predicted protein as mGlu1g receptor—the shortest of all mGlu1 receptor splice variants. The third mRNA, named mGlu1h, encodes a predicted C-terminal splice variant of 10 additional amino acids. mGlu1h mRNA was observed in two different melanoma cell lines and is overexpressed, compared with melanoma precursor cells, melanocytes. Most importantly, this new splice variant, mGlu1h receptor, is encoded by two previously unidentified exons located within the human GRM1 gene. Additionally, these new exons are found exclusively within the GRM1 genes of higher primates and are highly conserved. Therefore, we hypothesize that mGlu1h receptors play a distinct role in primate glutamatergic signaling.     

Acyl coenzyme A: cholesterol acyltransferase types 1 and 2: structure and function in atherosclerosis

Two enzymes are responsible for cholesterol ester formation in tissues, acyl coenzyme A:cholesterol acyltransferase types 1 and 2 (ACAT1 and ACAT2). The available evidence suggests different cell locations, membrane orientations, and metabolic functions for each enzyme. ACAT1 and ACAT2 gene disruption experiments in mice have shown complementary results, with ACAT1 being responsible for cholesterol homeostasis in the brain, skin, adrenal, and macrophages. ACAT1 -/- mice have less atherosclerosis than their ACAT1 +/+ counterparts, presumably because of the decreased ACAT activity in the macrophages. By contrast, ACAT2 -/- mice have limited cholesterol absorption in the intestine, and decreased cholesterol ester content in the liver and plasma lipoproteins. Almost no cholesterol esterification was found when liver and intestinal microsomes from ACAT2 -/- mice were assayed. Studies in non-human primates have shown the presence of ACAT1 primarily in the Kupffer cells of the liver, in non-mucosal cell types in the intestine, and in kidney and adrenal cortical cells, whereas ACAT2 is present only in hepatocytes and in intestinal mucosal cells. The membrane topology for ACAT1 and ACAT2 is also apparently different, with ACAT1 having a serine essential for activity on the cytoplasmic side of the endoplasmic reticulum membrane, whereas the analogous serine is present on the lumenal side of the endoplasmic reticulum for ACAT2. Taken together, the data suggest that cholesterol ester formation by ACAT1 supports separate functions compared with cholesterol esterification by ACAT2. The latter enzyme appears to be responsible for cholesterol ester formation and secretion in lipoproteins, whereas ACAT1 appears to function to maintain appropriate cholesterol availability in cell membranes.     

Calmodulin binding to the C-terminus of the small-conductance Ca2+-activated K+ channel hSK1 is affected by alternative splicing

We identified three splice variants of hSK1 whose C-terminal structures are determined by the independent deletion of two contiguous nucleotide sequences. The upstream sequence extends 25 bases in length, is initiated by a donor splice site within exon 8, and terminates at the end of the exon. The downstream sequence consists of nine bases that compose exon 9. When the upstream sequence (hSK1(-)(25b)) or both sequences (hSK1(-)(34b)) are deleted, truncated proteins are encoded in which the terminal 118 amino acids are absent. The binding of calmodulin to these variants is diminished, particularly in the absence of Ca2+ ions. The first 20 amino acids of the segment deleted from hSK1(-)(25b) and hSK1(-)(34b) contain a 1-8-14 Ca2+ calmodulin binding motif, and synthetic oligopeptides based on this region bind calmodulin better in the presence than absence of Ca2+ ions. When the downstream sequence (hSK1(-)(9b)) alone is deleted, only the three amino acids A452, Q453, and K454 are removed, and calmodulin binding is not reduced. On the basis of the relative abundance of mRNA encoding each of the four isoforms, the full-length variant appears to account for most hSK1 in the human hippocampus, while hSK1(-)(34b) predominates in reticulocytes, and hSK1(-)(9b) is especially abundant in human erythroleukemia cells in culture. We conclude that the binding of calmodulin by hSK1 can be modulated through alternative splicing.     

Alternative splicing and gene structure of the transforming growth factor beta-activated kinase 1

We have identified a fourth splice variant of the TGF beta-activated kinase (TAK1), called TAK1-d, and identified an error in the previously published TAK1-c sequence. Our data shows that the c and d variants encode proteins whose carboxyl ends differ markedly from those of variants a and b. Analysis of the human TAK1 gene sequence, located at 6q16.1-q16.3, shows that the coding sequence is organised in 17 exons. The four splice variants result from alternative splicing of exons 12 and 16, the reading frame of exon 17 being determined by the presence or absence of exon 16. Study of the relative levels of expression of the four splice variants showed significant variations between tissues. Our evidence suggests that the alternative splicing of the TAK1 mRNA may have important functional implications.     

Identification of putative active site residues of ACAT enzymes

In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.     

Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions

In order to further establish zebrafish as a vertebrate model for studying the mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity it is necessary to characterize the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AhR/ARNT) signaling pathways in this species. In this study, three zfARNT2 cDNAs were isolated, expressed, and characterized and named zfARNT2b, zfARNT2c, and zfARNT2a. zfARNT2b, zfARNT2c, and zfARNT2a encode proteins with theoretical molecular weights of 81, 79, and 45 kDa, respectively. zfARNT2b and zfARNT2a proteins are identical over the first 403 amino acids but differ in their C-terminal domains as a result of alternative mRNA splicing. zfARNT2c is nearly identical to zfARNT2b, with the exception of an in frame 15 amino acid deletion adjacent to the basic region of zfARNT2c. Using quantitative RT-PCR methods the tissue distribution of each zfARNT2 isoform was determined. In COS-7 cells expressing zfARNT2b and zfAhR2, 10 nM TCDD causes a nine-fold induction of a dioxin responsive reporter gene. In COS-7 cells expressing zfARNT2a or zfARNT2c, TCDD does not induce reporter gene expression. In contrast, all three zfARNT2 proteins induce reporter gene activity under control of hypoxia responsive elements when cotransfected with the zebrafish endothelial specific PAS protein 1. DNA gel shift analysis suggests that the decreased function of zfARNT2a is due to inefficient binding of zfARNT2a/zfAhR2 complexes to dioxin responsive elements. These results also indicate that alternative mRNA splicing results in formation of ARNT proteins with distinct functional properties.     

Molecular cloning of a splicing variant of human RECQL helicase

A cDNA was isolated from the human heart library. This cDNA variant was produced by the deletion of 176 bases at the 5(') end of human RecQL gene, presumably by an alternative mRNA splicing. The cDNA contains two open reading frames and so may encode two isoforms of human RECQL. The first isoform is a 105 amino acid protein with the first 53 N-terminal amino acids identical to the known sequence of RECQL protein and followed by 52 amino acids introduced by in-frame premature stop codon. The second isoform is a 537 amino acid protein that has the same sequence as the published human RECQL helicase, except the first 112 amino acids at the N-terminal end were absent. The possible roles of both of these proteins are discussed.