Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters

A cDNA that encodes a novel Na+-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na+-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters.     

Homologues of amino acid permeases: cloning and tissue expression of XAT1 and XAT2

The L-type (LAT) family of amino acid transporters is composed of exchangers for neutral, cationic, and anionic amino acids. They form functional heterodimers with membrane glycoproteins, rBAT or 4F2hc/CD98, to which they are linked by a disulphide bond. We report the molecular cloning and tissue expression of new mouse and human homologues of the LAT family, termed mXAT1, mXAT2 and hXAT2. The latter two proteins may correspond to ortholog genes in mouse and human. The hXAT2 gene is located on chromosome 8q21.3. The cloned X amino acid transporter (XAT) cDNAs are predicted to encode proteins of about 50 kDa. From a phylogenetic point of view, the three XAT proteins cluster together, but sequence comparison and secondary structure prediction show that they are also related to the members of the LAT family. Like these transporters, the XAT proteins show 12 transmembrane domains and a conserved cysteine residue, located in the second extracellular loop. This conserved cysteine is involved in the disulphide bond formed between the known members of the LAT family and 4F2hc or rBAT. The mXAT1 and hXAT2 mRNAs are expressed in the kidney but they are not detectable in a variety of other tissues. The corresponding proteins were efficiently translated following transfection of their cDNAs in Chinese hamster ovary (CHO) cells. However, cDNA transfection in CHO cells did not induce amino acid uptake, even when cotransfected with vectors expressing 4F2hc or rBAT. This could be related to the fact that mXAT1 and hXAT2 did not form detectable disulphide-linked heterodimers with 4F2hc or rBAT when they were co-expressed in CHO cells. Identification of other putative partner(s) of these LAT family-related transporters may be necessary to understand their role in renal physiology.     

牛Pbx-1基因的电子克隆及生物信息学分析

新基因全长cDNA序列很难获得,但电子克隆却提供了基因克隆的一种策略.利用小鼠Pbx-1基因编码序列(NM_183355)为种子序列进行电子克隆获得牛Pbx-1基因完整编码序列.然后,用生物信息学方法分析了牛的Pbx-1基因的结构,密码子偏性和氨基酸的同源性等.结果表明:该基因cDNA全长1 754 bp,无内含子,最大开放阅读框1 305 bp.编码434个氨基酸.预测其编码的蛋白分子量为47 189.5 Da,与小鼠的同源性为81%.     

牛Irak-1基因的电子克隆及生物信息学分析

电子克隆提供了一种利用基因组数据库克隆新基因全长cDNA序列的策略。利用小鼠Irak-1基因编码序列(NM_008363)为种子序列进行电子克隆获得了牛Irak-1基因完整编码序列。然后,用生物信息学方法分析了该基因的结构,微卫星位点,密码子偏性和氨基酸的同源性等。结果表明:该基因cDNA全长2 645bp,无内含子,最大开放阅读框2 157bp,编码718个氨基酸,与小鼠的同源性为77%。     

Identification of the tobacco and Arabidopsis homologues of the pollen-expressed LAT59 gene of tomato

We describe the complete genomic sequences for the tobacco and Arabidopsis homologues of tomato LAT59, a previously described member of a family of pectate lyase-like genes. Translation of the tobacco gene, Nt59, predicts a protein with 93.5% overall amino acid similarity to LAT59. Nt59 has two introns whose positions are exactly conserved with the two introns of LAT59. Both LAT59 and Nt59 are specifically expressed in pollen and their promoter and 5-UTR sequences are highly similar. Furthermore, two promoter elements shown to be important for pollen expression of LAT59 are conserved in the Nt59 promoter. The Arabidopsis homologue, At59, was found by examination of four candidates. At59 has 72.6% amino acid similarity to LAT59 and the position of one of its two introns is conserved with one of the LAT59 introns. At59 is also pollen-expressed and although its promoter sequence is quite different from the Nt59 and LAT59 promoters, the two promoter elements are somewhat conserved.     

日本脑炎病毒E基因与流感病毒HA基因的串联表达及应用

根据GenBank公布的日本脑炎病毒(Japanese Encephalitis Virus, JEV) SA14 14 2株的核酸序列和人流感病毒的血凝素基因(ha)序列, 设计一对特异性引物, 用 PCR方法扩增编码 JEV囊膜蛋白主要抗原域基因, 其中含ha基因主要核苷酸序列。将PCR产物定向克隆入原核表达载体 pET 32a( ), 构建原核表达载体 pET EHA。阳性质粒转化BL21(DE3)宿主菌, 经 IPTG诱导获得表达, 重组蛋白以包涵体的形式存在。Western blot分析表明表达产物具有良好的免疫学活性。利用纯化的表达产物与流感病毒血凝素单抗及乳胶建立了诊断日本脑炎病毒抗体水平的乳胶凝集试验。结果表明乳胶凝集方法具有简便快速、敏感性高、特异性强、价格低廉、可现场检测等优点, 是一种适合基层兽医单位用于日本脑炎病毒抗体水平检测的新方法。     

Cloning and functional characterization of a Na(+)-independent, broad-specific neutral amino acid transporter from mammalian intestine

We have isolated a cDNA from a rabbit intestinal cDNA library which, when co-expressed with the heavy chain of the human 4F2 antigen (4F2hc) in mammalian cells, induces system L-like amino acid transport activity. This protein, called LAT2, consists of 535 amino acids and is distinct from LAT1 which also interacts with 4F2hc to induce system L-like amino acid transport activity. LAT2 does not interact with rBAT, a protein with a significant structural similarity to 4F2hc. The 4F2hc/LAT2-mediated transport process differs from the 4F2hc/LAT1-mediated transport in substrate specificity, substrate affinity, tissue distribution, interaction with D-amino acids, and pH-dependence. The 4F2hc/LAT2-associated transport process has a broad specificity towards neutral amino acids with K(t) values in the range of 100-1000 microM, does not interact with D-amino acids to any significant extent, and is stimulated by acidic pH. In contrast, the 4F2hc/LAT1-associated transport process has a narrower specificity towards neutral amino acids, but with comparatively higher affinity (K(t) values in the range of 10-20 microM), interacts with some D-amino acids with high affinity, and is not influenced by pH. LAT2 is expressed primarily in the small intestine and kidney, whereas LAT1 exhibits a much broader tissue distribution.     

Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli.

Lysine epsilon-aminotransferase (LAT) in the beta-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a beta-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an approximately Mr 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies approximately 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the beta-lactam biosynthetic cluster between pcbC and cefE (approximately 25 kbp) is nearly complete.     

Identification of LAT4, a novel amino acid transporter with system L activity

System L amino acid transporters mediate the movement of bulky neutral amino acids across cell membranes. Until now three proteins that induce system L activity have been identified: LAT1, LAT2, and LAT3. The former two proteins belong to the solute carrier family 7 (SLC7), whereas the latter belongs to SLC43. In the present study we present a new cDNA, designated LAT4, which also mediates system L activity when expressed in Xenopus laevis oocytes. Human LAT4 exhibits 57% identity to human LAT3. Like LAT3, the amino acid transport activity induced by LAT4 is sodium-, chloride- and pH-independent, is not trans-stimulated, and shows two kinetic components. The low affinity component of LAT4 induced activity is sensitive to the sulfhydryl-specific reagent N-ethylmaleimide but not that with high affinity. Mutation in LAT4 of the SLC43 conserved serine 297 to alanine abolishes sensitivity to N-ethylmaleimide. LAT4 activity is detected at the basolateral membrane of PCT kidney cells. In situ hybridization experiments show that LAT4 mRNA is restricted to the epithelial cells of the distal tubule and the collecting duct in the kidney. In the intestine, LAT4 is mainly present in the cells of the crypt.     

大鼠脑红蛋白基因编码区的克隆、多态性分析及该基因组织表达谱研究

参考人和小鼠脑红蛋白（Neuroglobin,NGB)的cDNA序列设计简并引物,用RT－PCR方法从大鼠脑组织中扩增出大鼠NGB基因编码区的cDNA序列,该序列与小鼠NGB基因编码区的序列同尖性为96％,与人NGB基因编码区的序列同源性为88％,进一步分析表明,大鼠NGB基因编码区存在多个多态性位点;113t/c[138P],133a/g[N45D],388a/g[R130G],417t/c.该序列已被GenBank接受,登录号为AF333245,RT－PCR分析表明,该基因在大鼠脑,肝,肾,心肌和骨骼肌中均有较高水平表达,提示了其功能上的重要性。