中华鲟及六种淡水养殖鱼类脂蛋白脂酶和肝脂酶基因克隆及系统进化分析

为了研究鱼类脂蛋白脂酶(1iportein lipase,LPL)、肝脂酶(hepatic lipase,HL)基因结构、功能及分子系统关系,作者克隆了中华鲟(Acipenser sinensis)、鲢(Hypophthalmichthys molitrix)、鳙(Aristichthys nobilis)、草鱼(Ctenopharyngodon idellus)、鲮鱼(Cirrhinus molitorella)、尼罗罗非鱼(Oreochromis niloticus)和斑鳢(Channa maculata)的LPL和HL基因cDNA核心序列,并推测了其相应氨基酸序列.同时,还应用5'RACE和3'RACE技术分别扩增中华鲟、鲢肝脏LPL基因与中华鲟肝脏HL基因cDNA全序列.序列同源性分析表明,LPL和HL氨基酸序列分别在哺乳类动物、鸟类、鱼类中相对保守.与已知的脊椎动物内皮脂酶(endothelial lipase,EL)和胰脂酶(pancreatic 1ipase,PL)氨基酸序列构建系统进化树,发现LPL、HL、EL与PL同属脂肪酶家族,四者聚集成一有根树.     

Sequence of rat lipoprotein lipase-encoding cDNA.

A rat lipoprotein lipase (LPL)-encoding cDNA (LPL) has been entirely sequenced and compared to the sequences of all the LPL cDNAs reported in other species. As expected, high homology was found between the coding exons. The putative catalytic triad, Ser132, Asp156, His241, according to human numbering, is conserved in rat. As is the case in mouse, an Asn444 present in human LPL is also missing. The major divergences between human, mouse and rat LPLs were observed in the untranslated exon 10, where (i) the rat cDNA exhibits a 157-bp insertion and an 81-bp deletion relative to human; (ii) neither the B1 repeat nor the homopurine stretch reported in mouse can be recognized, and (iii) the rat cDNA displays several A+T-rich stretches.     

Expression of lipoprotein lipase associated with lung adenocarcinoma tissues

Lipoprotein lipase (LPL) plays a key role in the lipid metabolism and transporting. It can catalyze the hydrolysis of chylomicron and very low-density lipoprotein triglyceride. Moreover, the abnormality of LPL associates with many pathophysiological conditions. Herein cDNA microarray and Northern blots analysis were used to study the expression of lipoprotein lipase in lung adenocarcinoma tissues. There were 113 genes of all tested blots in cDNA microarray expressed lowly. LPL gene is expressed lowly at the average ratio 0.26 (Cy5/Cy3) in lung adenocarcinoma tissues over controls. Northern blots confirmed those changes detected from the cDNA microarray and suggested that low expression of LPL may play an important role in the lung adenocarcinoma development.     

Organization of the lipoprotein lipase gene of red sea bream Pagrus major

Lipoprotein lipase (LPL) is a key enzyme of lipid deposition and metabolism. To investigate the mechanism of lipid deposition in fish, as a first step, we have characterized the LPL gene of a marine teleost red sea bream Pagrus major by cDNA and genomic structure analysis. The red sea bream LPL gene encodes 511 amino acids and spans approximately 6.3 kb of the genome. The coding region is organized into ten exons and nine introns. In comparison with the LPL of other animals, the deduced amino acid sequence shows a high degree of similarity with a conservation of functional domains, e.g. catalytic triad, N-glycosylation sites, lipid and heparin binding regions. The 1.1 kb of 5′ flanking region contains two CCAAT, sequences homologous to Oct-I site and response elements for hormones including glucocorticoid, insulin and thyroid hormone. The results of the present study will facilitate further study of the function and regulation of the LPL in non-mammalian vertebrates.     

脂蛋白脂酶基因的克隆、序列测定及定点突变

 以人的脂肪组织总RNA为模板 ,参考已报道的脂蛋白脂酶 (lipoproteinlipase ,LPL)cDNA设计引物 ,利用RT PCR方法扩增得到了LPLcDNA ,并经序列测定证实其序列是正确的 .在冠心病患者LPL基因第 5外显子的 830位碱基处发现了G→A的转换 ,该变异导致LPL基因第 192位的密码子CGA被CAA取代 ,使LPL第 192位精氨酸改变为谷氨酰胺 .在变异碱基附近设计合成两条引物 ,其中一条包含所要改变的碱基 ,利用基于PCR的定点突变技术和体外重组的方法获得了G830A变异的LPLcDNA     

In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion

Detailed structure-function information about human lipoprotein lipase (LPL) is unavailable because it is difficult to purify large amounts of the enzyme for study. To circumvent this problem, we constructed an in vitro LPL expression vector. Human LPL cDNA was cloned and inserted into the expression vector p91023(B). After transfection of COS M-6 cells with the human LPL cDNA construct, LPL enzyme activity was detected in cell extracts and culture medium. Purified human apolipoprotein C-II caused a 5-fold stimulation of the recombinant human LPL expressed in vitro. Using site-specific mutagenesis, Ala residues were substituted for Asn residues at two potential N-linked glycosylation sites (positions 43 and 359) and at a third unrelated Asn (position 257) in the LPL cDNA. RNA blot analysis demonstrated the presence of a single mRNA species in COS cells transfected with wild-type and mutant LPL expression vectors. Intracellular and secreted LPL activity was absent in the construct containing an Ala for Asn mutation at position 43, whereas the same substitutions at positions 257 and 359 did not appreciably affect activity. LPL activity was also absent in another construct containing a Gln for Asn mutation at position 43. Quantitation of LPL protein mass concomitant with measurement of enzyme activity showed that substitution of Ala or Gln for Asn at position 43 resulted in the production of an enzymatically inactive protein which accumulated intracellularly but was not secreted into the culture medium. Our report represents an initial documentation of the expression of cloned human LPL in vitro and of the importance of Asn-43 for both enzyme activity and secretion.     

Transgenic rabbits expressing human lipoprotein lipase

To study the functions of lipoprotein lipase (LPL) in lipid and lipoprotein metabolism and the relationship between LPL and atherosclerosis, we generated transgenic rabbits expressing the human LPL gene. A total of 4045 Japanese whiterabbit embryos were microinjected with a 3.8-kb SalI/HindIII fragment containing the chicken -actin promoter, human LPL cDNA and rabbit -globin with poly (A) signals, and then transplanted into 116 recipient rabbits. Of the 166 pups born, six pups were transgenic as confirmed by Southern blot analysis. ANorthern blot analysis revealed that human LPL was expressed by a number of tissues including the heart, kidney, adrenal gland and intestine. One transgenic rabbit showed up to 3-foldincreased LPL activity in post-heparin plasma compared to thatin nontransgenic rabbits. Human LPL expression in various tissues of transgenic rabbits was further elucidated by in situ hybridization and immunostaining. Since rabbits are superior to mice as a model of atherosclerosis, this transgenicrabbit model should provide a valuable tool for the study of LPL in lipid metabolism and atherosclerosis.     

Role of A kinase anchor proteins in the tissue-specific regulation of lipoprotein lipase

Activation of protein kinase A by catecholamines inhibits lipoprotein lipase (LPL) activity through the elaboration of an RNA binding complex, which inhibits LPL translation by binding to the 3'-untranslated region of the LPL mRNA. To better define this process, we reconstituted the inhibitory RNA binding complex in vitro and demonstrated that the K homology (KH) domain of A kinase anchor protein (AKAP) 121/149 plays a vital role in the inhibition of LPL translation. Inhibition of LPL translation occurred in vitro only when the Calpha subunit, R subunit, and AKAP 149 were present. Using different glutathione-S-transferase fusion proteins of AKAP 149, sequences containing the KH domain were required for inhibition of LPL translation, and the inhibition of AKAP 121 expression in 3T3-F442A adipocytes with short interfering RNA resulted in loss of epinephrine-mediated translation inhibition. After epinephrine injection into mice, LPL activity was inhibited in white adipose tissue but not in brown adipose tissue (BAT) or muscle. LPL activity and synthetic rate were inhibited in vitro by the addition of epinephrine to 3T3-F442A adipocytes, but there was no effect in L6 muscle cells and cultures of brown adipocytes. Corresponding with these differences in LPL translation, AKAP 121 protein and mRNA were abundantly expressed in mouse white adipose tissue, but was either very low or undetectable in BAT and muscle. Thus, AKAP 121/149 contains a KH region that is essential to the translation inhibition of LPL in response to epinephrine. BAT and muscle do not express significant AKAP 121/149, and this likely explains some of the tissue-specific differences in LPL regulation.     

Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes

Lipoprotein lipase (LPL) is the enzyme responsible for hydrolysis of circulating triglyceride-rich lipoproteins and is important for storage of adipocyte lipid. To study the regulation of LPL synthetic rate in adipose tissue, primary cultures of isolated rat adipocytes were pulse-labeled with [35S]methionine, and LPL was immunoprecipitated with an LPL-specific antibody. A pulse-chase experiment identified the cellular and secreted forms of LPL as a 55-57-kDa protein. In the presence of heparin, there was a large increase in secretion of newly synthesized LPL from the cells, although heparin did not stimulate cellular LPL synthetic rate. When cells were exposed to insulin for 2 h, pulse-labeling revealed that insulin stimulated a maximal dose-related increase in LPL synthetic rate of 300% of control. This increase in LPL synthetic rate was observed after an exposure to insulin for as little as 60 min and was accompanied by only a 10-25% increase in total protein synthesis. In addition, insulin had no effect on the turnover of intracellular LPL. Using a cDNA probe for LPL, insulin induced a 2-fold increase in the LPL mRNA. Thus, insulin stimulated an increase in specific LPL mRNA in isolated rat adipocytes. This increase in LPL mRNA then leads to an increase in the synthetic rate of the LPL protein.     

海水鱼真鲷脂蛋白脂肪酶基因cDNA序列与组织表达

为研究脊椎动物真鲷脂蛋白脂肪酶 (LPL)结构与功能关系以及探讨动脉粥样硬化形成机理 ,通过构建cDNA文库 ,克隆对动脉粥样硬化表现抗性的海水鱼真鲷LPL基因cDNA全序列 .再通过PCR方法扩增基因组DNA ,获取内含子 9及其两侧序列以确定外显子 10的大小 ,最后通过RT PCR ,以 β肌动蛋白为外参照 ,比较真鲷在食用两种脂肪含量不同饲料和摄食状态不同的处理条件下 ,肝脏和腹腔肠系膜脂肪组织LPLmRNA的相对水平 .从腹腔肠系膜脂肪组织cDNA文库中克隆出LPLcDNA序列 ,其完整的开放阅读框架由 15 36bp组成 ,编码 5 11个氨基酸残基 .与哺乳类不同 ,真鲷LPL基因外显子 10的开始部分是翻译的 .LPL的催化位点、二硫键位点、N 糖基化位点、肝素结合区、脂质结合位点、介导脂蛋白与低密度脂蛋白受体结合位点、二聚体形成位点等主要功能域在真骨鱼类真鲷与其它脊椎动物间基本保守 ,但肝素结合区的碱性氨基酸残基含量较人类减少 ,并在结合脂质底物的疏水环套中出现插入片段 .与哺乳类不同 ,真鲷LPL基因在成体肝脏存在诱导性表达 ,而在其腹腔肠系膜脂肪组织则存在与哺乳类相似的组成性表达 .当真鲷喂食高脂饲料时 ,其饱食状态下肝脏LPLmRNA水平升高 ,但对其腹腔肠系膜脂肪组织LPL表达没有影响 .当真鲷喂食标准商业饲料时 ,     

Lipoprotein lipase (LPL) is highly expressed and active in the ovary of European sea bass (Dicentrarchus labrax L.), during gonadal development

The oocytes of many fish species accumulate high amounts of neutral lipids as a caloric reserve for embryonic and larval development. We propose that lipoprotein lipase (LPL, EC 3.1.1.34) plays an important role in supplying the oocytes with fatty acids and we have cloned its cDNA from the ovary of sea bass, and determined the patterns of LPL activity and LPL mRNA expression in the ovary. The cDNA obtained was 3051 bp long with an open reading frame encoding 518 amino acids. The amino acid sequence has a high similarity and shows similar structural features to LPL of other species. Northern blot analysis revealed LPL expression in adipose tissue and gonads only. LPL activity and LPL mRNA expression in the ovary was very high in fish with a gonadosomatic index (GSI) above 5, coinciding with the appearance of a high number of lipid droplets in the ooplasm. The LPL mRNA expression was localised to the follicle cells surrounding the oocyte. Our results suggest that LPL is likely to play an important role in the incorporation of neutral lipids into the oocytes, and that follicle cells, in addition to participating in steroidogenesis, also may be important in building up oocyte lipid reserves.     

An anti-ANGPTL3/8 antibody decreases circulating triglycerides by binding to a LPL-inhibitory leucine zipper-like motif

Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8. Remarkably, we found that LPL and ApoA5 both bound a unique ANGPTL3/8 epitope consisting of N-terminal regions of ANGPTL3 and ANGPTL8 that are unmasked upon formation of the ANGPTL3/8 complex. We further used ANGPTL3/8 as an immunogen to develop an antibody targeting this same epitope. After refocusing on antibodies that bound ANGPTL3/8, as opposed to ANGPTL3 or ANGPTL8 alone, we utilized bio-layer interferometry to select an antibody exhibiting high-affinity binding to the desired epitope. We revealed an ANGPTL3/8 leucine zipper-like motif within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region, suggesting the mechanism by which ApoA5 lowers TG is via competition with LPL for the same ANGPTL3/8-binding site. Supporting this hypothesis, we demonstrate that the anti-ANGPTL3/8 antibody potently blocked ANGPTL3/8-mediated LPL inhibition in vitro and dramatically lowered TG levels in vivo. Together, these data show that an anti-ANGPTL3/8 antibody targeting the same leucine zipper-containing epitope recognized by LPL and ApoA5 markedly decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition.     

Genetic and developmental regulation of the lipoprotein lipase gene: loci both distal and proximal to the lipoprotein lipase structural gene control enzyme expression

We report here a study of the developmental and genetic control of tissue-specific expression of lipoprotein lipase, the enzyme responsible for hydrolysis of triglycerides in chylomicrons and very low density lipoproteins. Lipoprotein lipase (LPL) mRNA is present in a wide variety of adult rat and mouse tissues examined, albeit at very different levels. A remarkable increase in the levels of LPL mRNA occurs in heart over a period of several weeks following birth, closely paralleling developmental changes in lipase activity and myocardial beta-oxidation capacity. Large increases in LPL mRNA also occur during differentiation of 3T3L1 cells to adipocytes. As previously reported, at least two separate genetic loci control the tissue-specific expression of LPL activity in mice. One of the loci, controlling LPL activity in heart, is associated with an alteration in LPL mRNA size, while the other, controlling LPL activity in adipose tissue, appears to affect the translation or post-translational expression of LPL. To examine whether these genetic variations are due to mutations of the LPL structural locus, we mapped the LPL gene to a region of mouse chromosome 8 using restriction fragment-length polymorphisms and analysis of hamster-mouse somatic cell hybrids. This region is homologous to the region of human chromosome 8 which contains the human LPL gene as judged by the conservation of linked genetic markers. Genetic variations affecting LPL expression in heart cosegregated with the LPL gene, while variations affecting LPL expression in adipose tissue did not. Furthermore, Southern blotting analysis indicates that LPL is encoded by a single gene and, thus, the genetic differences are not a consequence of independent regulation of two separate genes in the two tissues. These results suggest the existence of cis-acting elements for LPL gene expression that operate in heart but not adipose tissue. Our results also indicate that two genetic mutations resulting in deficiencies of LPL in mice, the W mutation on chromosome 5 and the cld mutation on mouse chromosome 17, do not involve the LPL structural gene locus. Finally, we show that the gene for hepatic lipase, a member of a gene family with LPL, is unlinked to the gene for LPL. This indicates that combined deficiencies of LPL and hepatic lipase, observed in humans as well as in certain mutant strains of mice, do not result from focal disruptions of a cluster of lipase genes.     

Novel site in lipoprotein lipase (LPL415;-438) essential for substrate interaction and dimer stability

LPL, like other lipases, has the ability to hydrolyze water-insoluble lipid substrates, but the mechanism is incompletely understood. We previously demonstrated a 22-amino acid loop in the amino-terminal domain of LPL to be essential for interaction with lipid substrates (Dugi, K. A., H. L. Dichek, G. D. Talley, H. B. Brewer, Jr., and S. Santamarina-Fojo. 1992. J. Biol. Chem. 267: 25086-25091) and mediation of substrate specificity (Dugi, K. A., H. L. Dichek, and S. Santamarina-Fojo. 1995. J. Biol. Chem. 270: 25396-25401). The carboxy-terminal domain, LPL415-438, contains two highly conserved hydrophobic stretches, and represents a candidate region for substrate interactions. Specific point mutations or deletion of the region between the hydrophobic stretches (LPL419-430) caused up to 90% selective loss of hydrolyzing activity against water-insoluble triolein, but not against water-soluble tributyrin, implicating a crucial function for LPL419-430 in the interaction with lipid substrates. In contrast, mutations introduced into the hydrophobic regions led to concomitant changes in tributyrin and triolein activities. The presence of an additional positive charge at position 416 yielded a gain of function mutant with 3-fold increased activity. This mutant was about three times more stable at 37 degrees C than wild-type LPL, suggesting an important role for the hydrophobic regions in LPL dimer stability. In summary, our data demonstrate that the carboxy-terminal region LPL415-438 plays an important role in both the interaction of LPL with lipid substrates and the stability of the LPL homodimer.     

Lipoprotein lipase and hepatic lipase mRNA tissue specific expression, developmental regulation, and evolution

Lipoprotein lipase (LPL) and hepatic lipase (HL) enzyme activities were previously reported to be regulated during development, but the underlying molecular events are unknown. In addition, little is known about LPL evolution. We cloned and sequenced a complete mouse LPL cDNA. Comparison of sequences from mouse, human, bovine, and guinea pig cDNAs indicated that the rates of evolution of mouse, human, and bovine LPL are quite low, but guinea pig LPL has evolved several times faster than the others. 32P-Labeled mouse LPL and rat HL cDNAs were used to study lipase mRNA tissue distribution and developmental regulation in the rat. Northern gel analysis revealed the presence of a single 1.87 kb HL mRNA species in liver, but not in other tissues including adrenal and ovary. A single 4.0 kb LPL mRNA species was detected in epididymal fat, heart, psoas muscle, lactating mammary gland, adrenal, lung, and ovary, but not in adult kidney, liver, intestine, or brain. Quantitative slot-blot hybridization analysis demonstrated the following relative amounts of LPL mRNA in rat tissues: adipose, 100%; heart, 94%; adrenal, 6.6%; muscle, 3.8%; lung, 3.0%; kidney, 0%; adult liver, 0%. The same quantitative analysis was used to study lipase mRNA levels during development. There was little postnatal variation in LPL mRNA in adipose tissue; maximal levels were detected at the earliest time points studied for both inguinal and epididymal fat. In heart, however, LPL mRNA was detected at low levels 6 days before birth and increased 278-fold as the animals grew to adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic organization of the region encoding guinea pig lipoprotein lipase; evidence for exon fusion and unconventional splicing

The coding sequence of guinea pig lipoprotein lipase (LPL) is organized into nine exons and spans a region of approximately 14 kb of the guinea pig genome. A non-conforming 5'-splice site is located on the first intron, which exhibits a 12-nucleotide perfect match with the 5'-end of the second exon. A previously described tryptic cleavage site is located on exon V, close to the 3' end of this exon. A similarity to vitellogenin resides on exons IV and V, and a putative active site is found on exon IV. A novel similarity to a fatty-acid-binding protein is noted on exon VI, adjacent to the postulated heparin-binding region. We suggest that free fatty acids (FFA) and heparin to some extent share the same site of interaction on the LPL molecule; and that a high local concentration of FFA can displace LPL from its site of action--the vascular endothelium--by competing for binding to heparan sulfate.