蛇毒C型凝集素类蛋白cDNA与基因组DNA序列分析显示特别的转录后加工_英文_

ISSN 　100727626 CN 　1123870ΠQ 中国生物化学与分子生物学报Chinese Journal of Biochemistry and Molecular Biology 2004 年 12 月20 (6) :713～718 Analysis of cDNAs and Genomic DNA of Snake Venom CTL2like Proteins Revealed An Extraordinary Post2transcriptional Processing Event ZHA Xiang2dong1) 3 , ZHOU Li2zhi1) , HUANG He2sheng2) , LIU Jing3) , XU Kang2sen4) (1) School of Life Sciences , Anhui University , Hefei 230039 , China ;2) Department of Pharmacology , Anhui Medical University , Hefei 230022 , China ; 3) School of Life Sciences , University of Science and Technology of China , Hefei 230026 , China ; 4) Institute for Control of Pharmaceuticals and Biological Products , Beijing 100050 , China) Abstract 　To better understand the accelerated evolution of snake venom C2type lectin2like proteins (CTL2like proteins) and to investigate the structure2function relationships , PCR was conducted to amplify cDNAs coding for theβchains of snake venom CTL2like proteins and the genomic DNA of agkisasinβ. The reaction products were cloned and sequenced. The causal relationships between the sequences and the experimental conditions were established. Dot plot analysis and multiple alignments were also performed. The results suggested the existence of a post2transcriptional processing event that was essentially homologous recombination at the RNA level , which might play an important role in the diversity of snake venom CTL2like proteins. This inference would provide a novel perspective for explaining a challenging problem of the post2genomic era : the discrepancy between the limited number of genes and the large collection of cDNAs , which was most prominent with regard to certain snake venom proteins. The genomic DNA of a snake venom C2type lectin2like protein was elucidated and no introns were found in the coding region. Key words 　C2type lectin2like proteins , intron ,post2transcriptional homologous recombination , accelerated evolution 蛇毒 C 型凝集素类蛋白 cDNA 与 基因组 DNA 序列分析显示特别的转录后加工 查向东1) 3 , 周立志1) ,黄河胜2) , 刘 　兢3) , 徐康森4) (1) 安徽大学生命科学学院 ,合肥　230039 ; 2) 安徽医科大学药理系 ,合肥　230022 ; 3) 中国科学技术大学生命科学学院 ,合肥　230026 ;4) 中国药品生物制品检定所 ,北京　100050) 摘要 　为研究蛇毒 C型凝集素类蛋白的快速进化机制和结构功能关系 ,使用 PCR 技术扩增了若干 编码 C型凝集素类蛋白β链的 cDNA 分子以及 agkisasinβ的基因组 DNA ,并将这些扩增产物进行克 隆和测序. 对测序结果与试验过程中的具体条件进行了因果关系分析 ,并且进行点阵图比较和多序 列比对. 结果表明 ,可能存在“转录后同源重组”等转录后的事件 ,在蛇毒 C 型凝集素类蛋白的多样 性上起着重要的作用. 对于解释基因数目与蛋白质数目的差异这一后基因组时代的重要问题 ,具有 一定的参考价值. 首次 报告 软件系统测试报告下载sgs报告如何下载关于路面塌陷情况报告535n,sgs报告怎么下载竣工报告下载 蛇毒 C型凝集素类蛋白的基因组 DNA 序列 ,其中未发现有内含子. 关键词 　C型凝集素类蛋白 ,内含子 ,转录后同源重组 ,快速进化 中图分类号 　Q781 1) Who contributed equally to this article 收稿日期 :2004202226 ,接受日期 :20042052103 联系人　Tel :055125106533 ;Fax :055125107354 ; E2mail :zhaxd @sina. com Received :February 26 ,2004 ;Accepted :May 10 ,20043 Corresponding author. Tel : 055125106533 ;Fxa :055125107354 ; E2mail :zhaxd @sina. com © 1994-2008 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net 　　Snake venom C2type lectin2like proteins ( CTL2like proteins) constitute a family of proteins that share high structural features and functionally interfere with haemostatic systems. The proteins all consist of two heterogeneous chains linked by an interchain disulfide bond each chain being structurally homologous to the carbohydrate2recognition domains (CRDs ) of animal C2 type lectins , therefore the proteins can be considered as members of C2type lectin superfamily and classified into group Ⅶ of C2type lectins[1 ] . However , the CTL2like proteins in snake venoms do not bind to carbohydrates as true lectins do and are not necessarily calcium dependent . Instead , their binding subjects are various proteins such as thrombin , factor vWF , coagulant factor Ⅸ, etc. Despite the highly similar structures , their biological activities and mechanism of actions are very distinct[2 ] . It is inferred that both chains of CTL2like proteins are evolved from C2type lectins , but during the long process of evolution they have developed different binding characteristics through alteration of some critical amino acid residues[1 ] , or created new binding properties through the interaction betweenα andβ subunits. The sequence alignment analysis revealed varying degree of identities (approximately from 30 % to 60 %) betweenα andβ chains of each CTL2like protein. Some deletions (or insertions) of one or several consecutive residues may account for the difference in their amino acid sequences , suggesting that the genes encoding the two chains are paralogous. The structures of mRNAs for the two chains of ⅨΠⅩ2bp have been elucidated , showing that the gene of each chain is transcribed and translated separately[3 ,4 ] . The number of CTL2like proteins (or cDNAs) found in snake venom continues to increase , and their structural diversity is prominent . Here we report several cDNAs coding forβchains of snake venom CTL2like proteins and the genomic DNA sequence of agkisasin β from Agkistrodon acutus venom , compared the sequences and also made cause and effect analysis between the sequence analysis results and the experimental conditions. Consequently we put forward our point of view about mechanisms underlying the accelerated evolution. 1 　Materials and Methods 1. 1 　Reagents Kits for RNA extraction , cDNA synthesis , plasmid purification and genomic DNA extraction were purchased from BioDev2Tech Co. Ltd , Beijing. ConcertTM DNA gel extraction system was purchased from Life Technologies. Taq DNA polymerase , T4 DNA ligase and Rnasin were from Sino2American Biotechnology Co. Plasmid pGEM2T Ò and E. coli strain JM109 competent cells were from Promega. 1. 2 　The snake venom protein sample The snake venom protein sample was purified and kindly provided by Professor Xiao Chang2hua from Kunming Zoology Institute. Its anticoagulant properties were identified while its sequence remained unknown. The 15 N2 terminus amino acid sequence , which would supply ground for designing PCR primers , was determined with Edman method on a 491 Protein Sequencer by the Sequencing Center in the School of Life Sciences ,Peking University. 1. 3 　RT2PCT and cDNA cloning 1. 3. 1 　Touchdown anchored PCR ( PCR1 ) and cDNA cloning of agkisasin β　Total RNA was isolated from venom gland of A. acutus originated from Hunan Province , and was reverse transcribed at 37 ℃for 2 hours with cDNA Synthesis Kit in which MuLV reverse transcriptase and random primer Oligo ( dT) 18 were included. We designed for PCR1 the degenerate upstream primer P1 : 5′2GATTGTCCXTCTGAYTGGTCCTCZTA23′ (X = A , C , T; Y= C , T; Z = C , T) , according to the first 15 N2terminal amino acid sequence of the snake venom protein : DCPSDWSSYEGHCYK, which was highly homologues with snake venom C2type lectin2like proteins as demonstrated by running Blastp program. Oligo (dT) 20 was used as the downstream primer. All PCR in the experiments were carried out in a Cyclogen Thermocycler ( Techne ) . The reaction products amplified with the touchdown anchored PCR method[5 ] was cloned into p GEM2T Ò and sequenced. In the selection plates containing ampicillin ( 100 μgΠml ) , seven separate positive colonies were picked in total and sent to Sangon Company and Takara Bio (Da Lian) Company respectively for sequencing. 1. 3. 2 　PCR amplification ( PCR2 ) and cloning of other cDNAs 　Because there exist numerous CTL2like proteins in snake venom and they share conserved amino acid sequences especially at the N2and2C termini , another RT2 PCR was performed to amplify cDNAs coding for those analogues. The same total RNA preparation was used as template in the reverse transcription. On the basis of the ORF of the cDNA sequence obtained above , a pair of specific primers was chosen : the upstream primer P1 , and the downstream primer P5 : 5′2TGCCT GGAACTCG CAGAC23′. Reaction volumes ( adjusted to 50 μl with ddH2O) comprised 115μl 10 mmolΠL dNTPs , 5μl 10 × PCR reaction buffer , 5μl 15 mmolΠL MgCl2 , 1μl Taq DNA polymerase (215 U) , the primers (011～015μmolΠ L) , and the template (10～500 ng) . Cycling parameters were as follows : 5 min at 95 ℃, followed by 28 cycles consisting of 30 s at 94 ℃, 60 s at 56 ℃, 60 s at 72 ℃ with a final extension for 5 min at 72 ℃. The amplified DNA was also cloned into p GEM2T Òand sequenced. 1. 4 　Amplif ication ( PCR3 ) and cloning of genomic DNA sequence Genomic DNA was extracted from the venom gland tissue of A . acutus . The A260ΠA280 ratio of the DNA 417 Chinese Journal of Biochemistry and Molecular Biology Vol. 20 © 1994-2008 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net preparation was 1181 , and its concentration was 36215 ngΠμl . To obtain genomic DNA sequence corresponding to the cDNA identified in Step 11311 , PCR was conducted with two primers designed also according to the 5′and 3′ terminal nucleotide sequences in the coding region of the cDNA : the forward primer P4 :5′2GATTGTCCTTCTGATT GGTCCT23′and the reverse primer P5 . The genomic DNA preparation was used as template in the catalytic reactions (about 700 ng of the genomic DNA preparation was taken for 50 μl reaction volume) . Other reaction conditions were the same as those of PCR2 except for setting varied annealing temperatures. The cloning procedures were as above2mentioned. 1. 5 　Software Primers were designed with the aid of Annhyb and Omiga 210. Multiple alignments of amino acid sequences and dot plot analysis were performed with Omiga 210. Results of multiple alignments were exported with Genedoc. Similarity search was performed by running BLAST program on http :ΠΠwww. ncbi . nlm. nih. gov. 2 　Results 2. 1 　cDNA sequence of agkisasinβ The touchdown anchored PCR and subsequent cDNA cloning resulted in the discovery of a single sequence possessing features of a cDNA , including an ORF coding for a peptide of 123 amino acids , and a 3′2UTR which contains a polyadenylation signal and a poly ( A ) +region. By running Blastp program , it was found that thededuced peptide was homologous to CRD of C2type lectinsand showed significant identity to theβ chains of othersnake venom CTL2like proteins and it was thusdenominated agkisasin β ( GenBank accession numberAF350324) . The result also implied that the reactionproducts in PCR1 were homogenous. Multiple2alignmentof agkisasinβwith its homologues from different speciesdemonstrated that identical fragments were more denselydistributed near N2termini than C2termini . As for theentire amino acid sequences , no 100 % sequence identitywas found among anyβchains.2. 2 　Cloning of cDNAs of analogues of agkisasinβand dot2plot analysisFour cDNAs were obtained in PCR2 with P1 and P5as specific primers and their deduced amino acidsequenced were denominated A , B , E and akitoninβ( GenBank accession number AF387100) . Despite theiroriginating from the same snake venom gland , thesequence variations indicated that the products of PCR2were heterogeneous. Furthermore , the dot plot analysissuggested a likely recombination between B and E thatgenerated A (Fig11) . Multiple alignment of A , B and E(Fig12) or their cDNAs ( Fig13) strongly supported theinference. Fig. 1 　An example of dot plot analysis among A , B and E ,the amino acid sequences deduced from cDNAs ofβchains of snake venom CTL2like proteins Fig. 2 　Comparison by multiple alignment of A , B and E , the amino acid sequences deduced from cDNAs ofβchains of snake venom CTL2like proteins 517No. 6 ZHA Xiang2dong et al :Analysis of cDNAs and Genomic DNA of Snake Venom CTL2like Proteins 　 © 1994-2008 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net Fig. 3 　Comparison by multiple alignment of A , B and E , cDNAs ofβchains of snake venom CTL2like Proteins 2. 3 　PCR amplif ication , cloning of genomic DNA sequence of agkisasinβ In PCR3 aimed at amplifying the genomic DNA agkisasin β, when the temperature for annealing was 53 ℃, several DNA fragments of different sizes ranging from 400 bp to 2 000 bp were displayed (Fig14) . Among them the fragment of about 1000 bp was cloned and sequenced. There was no apparent similarity between the entire deduced amino acid sequences and any other proteins by running Blastp progam. Nevertheless , some segments in the ORFs showed significant identity with microbial integrase and recombinase. Considering the accelerated evolution of some snake venom gland components , the existence of such mRNAs might be seen as signs of retrotransposition process leading to creation of newly found genes[6 ] . The value of this discovery remained to be further studied. To avoid the unspecific amplification we changed the PCR conditions by elevating the annealing temperature up to 56 ℃, then only the fragment of 400 bp was produced , Fig. 4 　Agarose gel electrophoresis of PCR amplification products The annealing temperature during thermo2cycles was 53 ℃ 1. PCR product ; 2. Molecular weight DNA marker which was cloned and sequenced. Sequencing results (Fig15) of several clones revealed that the genomic DNA sequences of agkisasinβ was almost identical with the cDNA of agkisasinβ, except that at the site 24 the base C is replaced by T (the sequence was accepted by GenBank and the accession number was AY293866) , which had no effect on the deduced amino acid sequence. This 617 Chinese Journal of Biochemistry and Molecular Biology Vol. 20 © 1994-2008 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net difference might be explained as individual variations since the venom glands used were from different snakes , which suggested gene polymorphism. The result definitely demonstrated that there was no intron in the genomic DNA sequence of agkisasinβ, at least in the region coding for the mature peptide. Fig. 5 　Genomic DNA sequence of agkisasinβ. The 24th base different from that in the cDNA was indicated by underlining 3 　Discussion 　　Although PCR1 featured a general primer Oligo (dT) 20 , a degenerate primer with a relatively high Tm value contrasting with the very low annealing temperature , the reaction products were homogenous ; while the products of PCR2 , in which both primers were specific and the annealing temperature was much higher , proved to be heterogeneous. It was abnormal that the stringent conditions rather resulted in non2specific amplification and vice versa . Another question could also be raised as to why only the nucleotide sequence of agkisasinβwas easily amplified in PCR1 and re2discovered in PCR3 but not in PCR2 . We inferred that the mRNAs for A ,B , E came from post2transcriptional processing including progressive truncation of poly(A) tail and recombination between the homologous mRNAs , and the shortened poly ( A) tails somehow hindered the amplification in PCR1 . The dot plot analysis and the multiple alignments seemingly support the RNA recombination hypothesis. A transitional consensus segment (from G184 to T197 in Fig. 3) might provide a clue in understanding the crossing2over mechanisms. As homologous recombination is generally precise and does not alter the length , it is also understandable that all β chains comprised 123 amino acids. The similar phenomenon was also observed in other CTL2like proteins[7 ] and in some snake venom thrombin2 like enzymes(data not shown) ,and could be interpreted in the same way. However ,our hypothesis is mainly based on the results of RT2PCR experiments and bioinformatic reasoning , which have certain limitations. Some other experiments , especially Northern blotting , should be conducted in future studies to gain a more profound insight and to help understand the mechanism. Many components in snake venoms , such as phospholipase A2 [8 ,9 ] , thrombin2like enzymes[10～12 ] , neurotoxins[13 ] , have also underwent accelerated evolution but the real mechanism remains unknown. For thrombin2 like enzymes , which have introns in the genomic structures , Siigur[11 ] attempted to explain the cDNA variation by alternative splicing , although sometimes it was impossible to do so because the splicing sites did not coincide with the known exon Π intron organizations. However , the genomic DNA of agkisasinβ does not have introns in the coding sequence , which implies that the structural diversity of snake venom CTL2like proteins cannot be explained with alternative RNA splicing , even in part . Our hypothesis has emphasized the contribution of post2transcriptional recombination to the diversity. Meanwhile , other mechanisms such as gene conversion , point mutation or RNA editing , may not be neglected. The DNA rearrangement in glandular cells may not yet be totally excluded and it is worthwhile to compare genomic sequences from different tissues of snakes. The accelerated evolution of snake venom components is a complicated phenomenon. A less stringent management of the genetic materials is probably the common feature , and different protein families may adopt different mechanisms or follow different evolutionary pathways. Lectins have been found in a lot of organisms. The diversity in their protein architectures is presumed to result from shuffling of exons encoding the CRD and other domains[14 ] . To our knowledge , this is also the first report about the genomic sequence of a snake venom C2type lectin2like protein , which does not contain introns. The CTL2like proteins may originate from retrotransposition of certain mRNAs , or the deletion2insertion of introns is a dynamic process[15 ] . Furthermore , we have constructed the phylogenetic trees for some snake venom thrombin2like enzymes and CTL2like proteins. The phylogenetic trees exhibited that thrombin2like enzymes were grouped in accordance with taxonomic units but the CTL2like proteins were not . Introns may play a critical role in maintaining the gene integrity or specificities by repelling homologous recombination with exogenous genes at DNA level . Acknowledgement 　The authors thank Dr. Yu Feng2an for his valuable advice and critical reading of the manuscript , and Prof . Wang Yu2zhen for her helps with reference to molecular evolution theory. References 1 　Drickamer K. C2type lectin2like domains. Curr Opin Struct Biol ,1999 , 9 : 585～590 2 　Cheng X , Qian Y W , Liu Q D , Li B X Y, Zhang M W , Liu J . Purification , characterization ,and cDNA cloning of a new fibrinogenlytic venom protein , agkisacutacin , from Agkistrodon acutus venom. Biochem Biophys Res Commun , 1999 ,265 :530～535 3 　Matsuzaki R , Yoshiara E , Yamada M , Shima K, Atoda H , Morita H. cDNA cloning of IXΠX2BP , a heterogenous two2chain anticoagulant 717No. 6 ZHA Xiang2dong et al :Analysis of cDNAs and Genomic DNA of Snake Venom CTL2like Proteins 　 © 1994-2008 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net protein from snake venom. Biochem Biophys Res Commun , 1996 , 220 : 382～387 4 　余红秀 ,向开军 ,汪玉洁 ,刘兢. 皖南尖吻蝮蛇中 11 个 C 型凝集 素类似蛋白亚单位的 cDNA 克隆和序列分析. 中国生物化学与 分子生物学报 ( Yu Hong2xiu ,Xiang Kai2jun ,Wang Yu2jie ,Liu Jing. Cloning and sequencing of eleven cDNAs of C2type lectin2like protein subunits from Agkistrodon acutus . Chin J Biochem Mol Biol) ,2003 ,19 (1) :64～70 5 　查向东 ,张华远 ,肖亚中 ,刘兢 ,徐康森. 尖吻蝮蛇未知 C 类凝集 素蛋白 cDNA 扩增、克隆与表达. 生物化学与生物物理进展 ( Zha Xiang2dong ,Zhang Hua2yuan , Xiao Ya2zhong ,Liu Jing , Xu Kang2sen. Amplifying ,cloning and expression of the cDNA encoding an unknown C2type lectin superfamily protein from Agkistrodon acutus . Prog Biochem Biophys) ,2001 ,28 (5) :752～755 6 　Courseaux A , Nahon J L. Birth of two chimeric genes in the Hominidae lineage. Science ,2001 , 291 (5507) :129321297 7 　Zha Xiang2dong , Liu Jing , Xu Kang2sheng. cDNA cloning , sequence analysis and recombinant expression of akitonin beta , a C2type lectin2 like protein from Agkistrodon acutus . Acta Pharmacol Sin , 2004 , 25 (3) :372～377 8 　Motonori O , Takahito C , Naoko O , Tomohisa O , Shosaku H. Molecular evolution of myotoxic phospholipases A2 from snake venom. 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