Novel gene exon homologous to pancreatic phospholipase A2: sequence and chromosomal mapping of both human genes

We described previously the cloning and DNA sequence of the human gene encoding pancreatic phospholipase A2 [DNA 5, 519]. When pancreatic phospholipase A2 (PLA2) cDNA was used to screen a human genomic library, two classes of clones were obtained. One class encoded the pancreatic enzyme, and a second class encoded one exon of an apparently related PLA2. No additional PLA2 gene exons displayed sufficient homology to be detected by the probe. A homologous sequence in both rat and porcine genomic DNA was detected by DNA blot hybridization, and the corresponding gene fragments were cloned and sequenced. Within the deduced amino acid sequences, the presence of known functional residues along with the high degree of interspecies conservation suggests the genes encode a functional PLA2 enzyme form. The encoded sequence lacks Cys11, as do the "type II" viperid venom and other nonpancreatic mammalian PLA2 enzymes. The sequence is distinct from porcine intestinal PLA2 and appears not to be a direct homolog of the recently published rabbit ascites and rat platelet enzymes. Hybridization of DNA probes containing sequences from these genes to genomic DNA blots of mouse/human somatic cell hybrids permitted chromosomal assignment for both. The pancreatic gene mapped to human chromosome 12, and the homologous gene mapped to chromosome 1.     

江浙蝮蛇磷脂酶A2基因的多样性研究

我们利用简并引物从江浙蝮蛇腺总ＲＮＡ经ＲＰ－ＰＣＲ扩增磷脂酶Ａ２（简称ＰＬＡ２）基因,并以碱性ＰＬＡ２（Ｂ－ＰＬＡ２）基因为探针,分离出了酸性ＰＬＡ２（Ａ－ＰＬＡ２）和两个未见报道的特征结构类同的基因,分别命名为Ａｓｎ＾４８－ＰＡＬ２和ＢＡ－ＰＡＬ２。双向测序测定了这组ＰＬＡ２同工酶（除信号肽外）基因的全序列,并由此推导编码的氨基酸序列。其中Ａ－ＰＬＡ２基因编码的氨基酸序列与较早报道的由蛇毒中分离     

Renaturation of cobra venom phospholipase A2 expressed from a synthetic gene in Escherichia coli.

Cobra venom (Naja naja naja) phospholipase A2 (PLA2) contains 14 cysteines in the form of 7 disulfide bonds amongst its 119 amino acids. A gene encoding the PLA2 was synthesized and inserted into a bacterial expression vector containing the phage lambda pL promoter. In order to obtain protein without the initiating methionine at the N-terminus, a Factor Xa site was engineered upstream from the PLA2 gene. Upon heat-induction of the cells transformed with the expression plasmid, the protein is produced as insoluble inclusion bodies. The enzyme was partially purified by washing the inclusion bodies with Triton X-100 and urea. The expressed protein was first denatured with 8 M guanidine-HCl and 10 mM DTT. After digestion with Factor Xa, formation of disulfide bonds and refolding into the fully active form was carried out in the presence of cysteine and Ca2+. The renatured recombinant protein was purified by Affi-gel blue column chromatography. The purified recombinant enzyme had the same specific activity as the native enzyme when assayed on a variety of substrates and cross-reacted with antisera prepared against the native enzyme. This is the first report of the expression of a recombinant PLA2 from any venom.     

Porcine pancreatic phospholipase A2 stimulates secretin release from secretin-producing cells

We have isolated, from canine pancreatic juice, two 14-kDa proteins with secretin-releasing activity that had N-terminal sequence homology with canine pancreatic phospholipase A2 (PLA2). In this study we have obtained evidence that secretin-releasing activity is an intrinsic property of pancreatic PLA2. Porcine pancreatic PLA2 from Sigma or Boehringer Mannheim was fractionated into several peaks by reverse phase high performance liquid chromatography. They were tested for stimulation of secretin release from murine neuroendocrine intestinal tumor cell line STC-1 and secretin cells enriched mucosal cell preparations isolated from rat upper small intestine. Each enzyme preparation was found to contain several components of secretin-releasing activity. Each bioactive fraction was purified to homogeneity by rechromatography and then subjected to mass spectral analysis and assays of PLA2 and secretin-releasing activities. It was found that the fraction with highest enzymatic activity also had the highest secretin-releasing activity and the same Mr as porcine pancreatic PLA2. Moreover, it also had the same N-terminal amino acid sequence (up to 30 residues determined) as that of porcine pancreatic PLA2, suggesting that it was identical to the enzyme. Purified porcine pancreatic PLA2 also stimulated secretin release concentration-dependently from both STC-1 cells and a mucosal cell preparation enriched in secretin-containing endocrine cells isolated from rat duodenum. Abolishment of the enzymatic activity by pretreatment with bromophenacyl bromide did not affect its secretin-releasing activity. The stimulatory effect of purified pancreatic PLA2 on secretin secretion from STC-1 cells was inhibited by an L-type Ca2+ channel blocker, by down-regulation of protein kinase C or by pretreatment of the cell with pertussis toxin. It is concluded that porcine pancreatic PLA2 possesses an intrinsic secretin-releasing activity that was independent of its enzymatic activity. This action is pertussis toxin-sensitive and is in part dependent on Ca2+ influx through the L-type channel and activation of protein kinase C.     

Molecular cloning of murine 72-kDa type IV collagenase and its expression during mouse development.

We report the isolation of a cDNA clone providing the first and complete sequence of mouse 72-kDa type IV collagenase. The clone contains 2800 nucleotides with a 1986-nucleotide open reading frame coding for 662 amino acids. The amino acid sequence includes a 29-residue signal peptide, an 80-residue propeptide, and a 553-residue enzyme proper. The sequence identity between the mouse and human enzymes is 96% with all cysteine residues conserved. The carboxyl-terminal domain of the mouse enzyme contains two more residues than the human enzyme. Northern hybridization analysis revealed considerable expression of the enzyme gene in newborn mouse lung, heart, kidney, and psoas muscle tissues, whereas only weak or no signals were observed in liver, spleen, and brain. Expression of the gene was substantially reduced in the same tissues of 3-month-old mice. In situ hybridization analysis of 72-kDa type IV collagenase expression in 10-15-day-old mouse embryos showed that the gene was intensely expressed in mesenchymal cells. Brain and surface ectoderm were completely negative. The epithelial tissue component of developing organs was negative with the exception of salivary gland. Although the expression varied somewhat between different mesenchymal tissues, no temporal or spatial changes could be associated with the advancement of epithelial branching morphogenesis. These findings together with our previous data on the expression of 72-kDa type IV collagenase in human tumors indicate that this enzyme has some very specific roles both in the physiological and pathological degradation of extracellular matrix. Furthermore, it has become clear that the closely related 92-kDa type IV collagenase differs completely with respect to expression pattern as well as gene regulation. The mouse cDNA clones reported in this study may provide important tools unraveling the actual roles of these enzymes in vivo.     

Cloning of mouse Dab2ip gene, a novel member of the RasGTPase-activating protein family and characterization of its regulatory region in prostate

Disabled homolog 2 (Drosophila) interacting protein (DAB2IP/Dab2IP) is a member of the GTPase-activating protein for downregulating the Ras-mediated signal pathway and TNF-mediated apoptosis. The downregulation of human DAB2IP mRNA levels was detected in prostate cancer cells due to the epigenetic regulation. Here, we isolated a mouse Dab2ip gene with a highly homologous sequence to that of the human and rat gene and mapped it at chromosome 2B. The mDab2ip gene contains 14 exons and 13 introns and spans approximately 65 kb. Exon1 contains at least three splicing variants (Ia, Ib, and Ic). The deduced amino acid sequence of mouse Dab2IP encompasses 1065 residues containing several unique protein interaction motifs as well as a Ras-like GAP-related domain, which shares a high homology with both humans and rats. Data from real-time RT-PCR analysis revealed a diverse expression pattern of the mDab2ip gene in various organs, implying differential regulation of this gene from various tissues. We have mapped a 1.3-kb segment containing a 5'-upstream region from exon Ia as a promoter region (-147/+545) in prostatic epithelial cell lines (TRAMP-C); this region is highly GC-rich, and mDab2ip appears to be a TATA-less promoter. It appears that epigenetic regulation, particularly histone acetylation of the Dab2ip gene promoter, plays an important role in modulating its gene expression in the mouse prostate cancer cell.     

Characterization of the D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate

The murine gene for the glucuronyl C5-epimerase involved in heparan sulfate biosynthesis was cloned, using a previously isolated bovine lung cDNA fragment (Li, J.-P., Hagner-McWhirter, A., Kjellén, L., Palgi, J., Jalkanen, M., and Lindahl, U. (1997) J. Biol. Chem. 272, 28158-28163) as probe. The approximately 11-kilobase pair mouse gene contains 3 exons from the first ATG to stop codon and is localized to chromosome 9. Southern analysis of the genomic DNA and chromosome mapping suggested the occurrence of a single epimerase gene. Based on the genomic sequence, a mouse liver cDNA was isolated that encodes a 618-amino acid residue protein, thus extending by 174 N-terminal residues the sequence deduced from the (incomplete) bovine cDNA. Comparison of murine, bovine, and human epimerase cDNA structures indicated 96-99% identity at the amino acid level. A cDNA identical to the mouse liver species was demonstrated in mouse mast cells committed to heparin biosynthesis. These findings suggest that the iduronic acid residues in heparin and heparan sulfate, despite different structural contexts, are generated by the same C5-epimerase enzyme. The catalytic activity of the recombinant full-length mouse liver epimerase, expressed in insect cells, was found to be >2 orders of magnitude higher than that of the previously cloned, smaller bovine recombinant protein. The approximately 52-kDa, similarly highly active, enzyme originally purified from bovine liver (Campbell, P., Hannesson, H. H., Sandb?ck, D., Rodén, L., Lindahl, U., and Li, J.-P. (1994) J. Biol. Chem. 269, 26953-26958) was found to be associated with an approximately 22-kDa peptide generated by a single proteolytic cleavage of the full-sized protein.     

Sequence specific inhibition of human type II phospholipase A2 enzyme activity by phosphorothioate oligonucleotides.

Phosphorothioate oligonucleotides were identified which directly inhibited human type II phospholipase A2 (PLA2) enzyme activity in a sequence specific manner. The minimum pharmacophore common to all oligonucleotides which inhibited PLA2 enzyme activity consisted of two sets of three or more consecutive guanosine residues in a row. These oligonucleotides appear to form G quartets resulting in the formation of oligonucleotide aggregates. Additionally, a phosphorothioate backbone was required to be effective inhibitors of type II PLA2. The activity of one oligodeoxynucleotide, IP 3196 (5'-GGGTGGGTATAGAAGGGCTCC-3') has been characterized in more detail. IP 3196 inhibited PLA2 enzyme activity when the substrate was presented in the form of a phospholipid bilayer but not when presented in the form of a mixed micelle with anionic detergents. Human type II PLA2 was 50-fold more sensitive to inhibition by IP 3196 than venom and pancreatic type I enzymes. These data demonstrate that phosphorothioate oligonucleotides can specifically inhibit human type II PLA2 enzyme activity in a sequence specific manner.     

大鼠睾丸特异表达基因Ube1的分离鉴定及生物学特征

本研究采用抑制性消减杂交(suppression subtracfive hybridization, SSH)和cDNA快速扩增(rapid amplification of cDNA ends, RACE)技术从大鼠A型精原细胞和粗线期精母细胞中成功克隆出大鼠泛素激活酶(ubiquitin-activating enzyme)基因Ube1 (GenBank登录号EF690356).该基因序列全长3433 bp,其中开放阅读框有3171 bp,编码一个含1057个氨基酸的蛋白质.Blast比对显示,Ube1与小鼠泛素激活酶基因Ubely1的同源性为93%,与人泛素激活酶基因UBE1的同源性为82%.Ube1基因编码的蛋白质含泛素激活酶信号位点和泛素激活酶活化位点,这些位点也存在于人类和小鼠的泛素激活酶1中.RT-PCR分析显示,Ube1在睾丸中大量表达,而在心、肝、脾、肺、肾、肌肉、脑、卵巢中没有表达.荧光定量PCR分析不同生精细胞中Ube1的表达,显示Ube1在A型精原细胞中大量表达,在粗线期精母细胞、圆形精子细胞和支持细胞中微弱表达.以上结果提示,Ube1是大鼠睾丸特异表达基因,可能通过参与泛素/蛋白酶体途径来影响精子发生.     

cDNA cloning, expression and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene(1).

Cytosolic thioredoxin (Trx) and thioredoxin reductase (TrxR) comprise a ubiquitous system that uses the reducing power of NADPH to act as a general disulfide reductase system as well as a potent antioxidant system. Human and rat mitochondria contain a complete thioredoxin system different from the one present in the cytosol. The mitochondrial system is involved in the oxidative stress protection through a mitochondrial thioredoxin-dependent peroxidase. We report here the cDNA cloning and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene (TrxR2). The mouse TrxR2 cDNA encodes for a putative protein of 527 amino acid residues with a calculated molecular mass of 57 kDa, that displays high homology with the human and rat counterparts. The N-terminus of the protein displays typical features of a mitochondrial targeting sequence with absence of acidic residues and abundance of basic residues. Mouse TrxR2 also contains a stop codon in frame at the C-terminus of the protein, necessary for the incorporation of selenocysteine that is required for enzymatic activity. The typical stem-loop structure (SECIS element) that drives the incorporation of selenocysteine is identified in the 3'-UTR. Northern analysis of the mouse TrxR2 mRNA shows a similar pattern of expression with the human homologue, with higher expression in liver, heart and kidney. Finally, we have assigned the mouse TrxR2 gene to chromosome 16 mapping at 11.2 cM from the centromer and linked to the catechol-o-methyltransferase (comt) gene.     

A strategy for obtaining active mammalian enzyme from a fusion protein expressed in bacteria using phospholipase A2 as a model

An active preparation of human phospholipase A2 (PLA2) was made after expression as an insoluble fusion protein in Escherichia coli. The new key elements required for PLA2 isolation were the maintenance of the fusion protein in solution after the initial solubilization and the use of a tryptophan cleavage procedure for regeneration of native PLA2 from the fusion protein. The fusion protein was composed of a beta-galactosidase leader peptide incorporating six consecutive threonine residues to aid in insoluble inclusion body formation, followed by a tryptophan adjacent to the N-terminus of PLA2. The fusion protein was purified from cell lysates, and the leader peptide was cleaved on the C-terminal side of the tryptophan residue with N-chlorosuccinimide. The released PLA2 was refolded and renatured to produce an enzyme with activity comparable to that of other phospholipases A2.     

The structural and functional contribution of N-terminal region and His-47 on Taiwan cobra phospholipase A2.

Modification of His-47 and removal of the N-terminal octapeptide caused a different effect on the structure of Naja naja atra (Taiwan cobra) phospholipase A2 (PLA2). Unlike native enzyme, Ca2+ induced an alteration in the structural flexibility of His-modified PLA2. Moreover, the spatial positions of Trp residues in His-modified PLA2 were not properly rearranged toward lipid-water interface in the presence of Ca2+. CD spectra and fluorescence measurement showed that the dynamic properties of Trp residues and the gross conformation of N-terminally truncated PLA2 were totally different from native enzyme. Although a precipitous drop in the enzymatic activity was observed with modified PLA2, His-modified PLA2 and N-terminally truncated PLA2 retained cytotoxicity on inducing necrotic death of human neuroblastoma SK-N-SH cells. Our data suggest that structural perturbations elicited by the chemical modification cause a dissociation of enzymatic activity and cytotoxicity of PLA2.