Sex- and tissue-specific expression of aspartic proteinases in Danio rerio (zebrafish)

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT–PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.     

Oestrogen-induced expression of a novel liver-specific aspartic proteinase in Danio rerio (zebrafish)

Aspartic proteinases are a group of endoproteolytic proteinases active at acidic pH and characterized by the presence of two aspartyl residues in the active site. They include related paralogous proteins such as cathepsin D, cathepsin E and pepsin. Although extensively investigated in mammals, aspartic proteinases have been less studied in other vertebrates. In a previous work, we cloned and sequenced a DNA complementary to RNA encoding an enzyme present in zebrafish liver. The sequence resulted to be homologous to a novel form of aspartic proteinase firstly described by us in Antarctic fish. In zebrafish, the gene encoding this enzyme is expressed only in the female liver, in contrast with cathepsin D that is expressed in all the tissues examined independently of the sex. For this reason we have termed the new enzyme liver-specific aspartic proteinase (LAP).Northern blot analyses indicate that LAP gene expression is under hormonal control. Indeed, in oestrogen-treated male fish, cathepsin D expression was not enhanced in the various tissues examined, but the LAP gene product appeared exclusively in the liver. Our results provide evidence for an oestrogen-induced expression of LAP gene in liver. We postulate that the sexual dimorphic expression of the LAP gene may be related to the reproductive process.     

家蚕组织蛋白酶D基因的克隆、序列分析及其表达谱研究

组织蛋白酶D (cathepsin D,CtD)是溶酶体内天冬氨酸内切蛋白酶,参与机体多种生理病理过程,尤其在昆虫的发育变态过程中起着重要作用。利用NCBI上登录的组织蛋白酶D基因核酸序列和家蚕Bombyx mori表达序列标签（expressed sequence tags, EST）数据库,进行电子克隆获得家蚕组织蛋白酶D (BmCtD) 基因的全长cDNA (DQ010007)。该cDNA大小为1 543 bp,其中ORF长1 152 bp,同源性分析表明BmCtD与其他物种的CtD具有较高的相似性。BmCtD的mRNA存在选择性拼接,另外一种mRNA形式命名为BmCtDⅠ。RT-PCR实验表明该基因在本实验所调查的家蚕不同发育时期和组织中都有表达。     

Molecular cloning, characterization and expression analysis of cathepsin D gene from turbot Scophthalmus maximus

Cathepsin D is a lysosomal endoproteolytic aspartic proteinase which also has been found in endosomes of macrophage. It is thought to play key roles in the developmental and physiological process of animals. The EST sequence of turbot (Scophthalmus maximus L.) cathepsin D was obtained from a subtractive cDNA library. In the present study, 5'-RACE and 3'-RACE were carried out to obtain the complete cDNA sequence of turbot cathepsin D, which contained a 91 bp 5'-UTR, a 1191 bp open reading frame encoding 396 amino acids, and a 329 bp 3'-UTR. The deduced amino acid sequence of the cathepsin D consisted of a signal peptide of 18 aa, a leader peptide extending 43 aa, and a mature peptide of 335 aa. BLAST analysis revealed that turbot cathepsin D shared high similarity with other known cathepsin D, and it showed significant homology with that of Barramundi (Lates calcarifer B., 89% aa similarity). Quantitative real-time PCR (q PCR) demonstrated that the highest expression level of the turbot cathepsin D was in liver. After turbot were challenged with Vibrio harveyi, the lowest expression levels of cathepsin D in liver, spleen and head kidney were detected at 8 h. This result was different from the expression of MHCII of which the expression lever was increased upon challenge. The expression levels of cathepsin D in liver and head kidney increased gradually after 8 h and exceeded the background level after 24 h. In spleen, the expression level was reinforced after 8 h and kept at level that was higher than the original level after 12 h. The results suggested that cathepsin D might process antigens for presentation to the immune system and have synergetic effect with apoptosis pathway until 12 h after injection.     

Characterization of mouse cathepsin R, a new member of a family of placentally expressed cysteine proteases

A new mouse cysteine protease, termed cathepsin R, has been identified. The complete nucleotide sequence of this gene was derived from a set of cDNAs generated from 15.5-day mouse placenta. Sequence analysis revealed an open reading frame encoding a 334 amino acid long polypeptide closely related to placentally expressed cathepsins P, Q, and M. RT-PCR analysis indicated that cathepsin R is only expressed in placenta and thus is a new member of the emerging family of cathepsins whose expression is regulated during mouse embryonic development. Modeling and structural analysis suggests that cathepsin R will have a restricted substrate specificity when compared to that of cathepsin L.     

Genomic organization of the Schistosoma mansoni aspartic protease gene, a platyhelminth orthologue of mammalian lysosomal cathepsin D

Schistosomes are considered the most important of the helminth parasites of humans in terms of morbidity and mortality. Schistosomes employ proteolytic enzymes to digest host hemoglobin from ingested human blood, including a cathepsin D-like, aspartic protease that is overexpressed in the gut of the adult female schistosome. Because of its key role in parasite nutrition, this enzyme represents a potential intervention target. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cathepsin D gene locus of Schistosoma mansoni. Using the cDNA encoding S. mansoni cathepsin D as a probe, we isolated several positive bacterial artificial chromosomes (BAC) from a BAC library that represents an approximately 8-fold coverage of the schistosome genome. Sequencing of BAC clone 25-J-24 revealed that the cathepsin D gene locus was approximately 13 kb in length, and included seven exons interrupted by six introns. The exons ranged in length from 49 to 294 bp, and the introns from 30 to 5025 bp. The genomic organization of schistosome cathepsin D was similar in sequence, structure and complexity to human cathepsin D, including to a greater or lesser extent the conservation of all six exon/intron boundaries of the schistosome gene. It was less similar to aspartic protease genes of the nematodes Caenorhabditis elegans and Haemonchus contortus, and dissimilar to those of plasmepsins from malarial parasites. Examination of the introns revealed the presence of endogenous mobile genetic elements including SR2, the ASL-associated retrotransposon, and the SINE-like element, SMalpha. Phylogenetically, schistosome cathepsin D appeared to be more closely related to mammalian cathepsin D than to other sub-families of eukaryotic aspartic proteases known from mammals. Taken together, these features indicated that schistosome cathepsin D is a platyhelminth orthologue of mammalian lysosomal cathepsin D.     

Differences in cathepsin B mRNA levels in rat tissues suggest specialized functions

The tissue distribution of mRNAs encoding two lysosomal proteases, cathepsin B and cathepsin D, was examined using cloned cDNAs to probe Northern and dot blots of RNAs extracted from various rat tissues. Cathepsin B mRNA showed a wide range of variation in expression in the tissues analyzed with the highest concentrations found in spleen and kidney, while the cathepsin D mRNA levels were relatively uniform in these same tissues. Significant quantities of cathepsin B mRNA were detected in total RNA from isolated islets of Langerhans but was not detectable in equivalent amounts of RNA from whole pancreas. The wide variations in tissue levels of cathepsin B mRNA suggest that tissue specific controls may regulate its expression and are compatible with the participation of this protease in specialized cellular functions other than intralysosomal protein degradation.     

人甲状腺受体相互作用蛋白15基因全长cDNA的克隆及其特性

通过生物信息学分析及RT PCR技术 ,从人垂体cDNA文库中克隆到甲状腺素受体相互作用蛋白 15(hTRIP15)的全长cDNA ,长度 1963bp ,编码 4 4 3氨基酸 ,同时克隆该基因不同剪接方式所形成的新的异构体 ,长度 1984bp ,编码 4 50氨基酸 .与基因组序列比较显示该基因具有 12个外显子 ,5号外显子 3′端具有 2个剪切的接点 (-ag) .搜寻UniGene数据库作染色体定位于D15S146 D15S117,该基因在生物进化上具有较高的保守性 ,从单细胞藻类到人类均有该基因同源物表达 ,亚细胞定位为核内 .Northern杂交显示 ,该基因具有 3种不同大小的转录本 ,分别约为 2 0、3 5及 4 0kb ,且在人体各组织中均有一定表达 ,其中骨骼肌、心脏及肾脏组织为高表达 .半定量RT PCR显示在一些内分泌组织均有表达 ,以肾上腺较高 .     

家蚕组织蛋白酶基因家族的鉴定及表达特征分析

家蚕是鳞翅目完全变态昆虫,在其变态过程中伴随着巨大的形态变化,包括旧组织的解离和新组织的形成,在这过程中有多种组织蛋白酶参与。组织蛋白酶是一类细胞内蛋白酶,广泛存在于各个物种中,包括组织蛋白酶B、H、L等几个亚家族。对家蚕组织蛋白酶的研究将有利于阐明家蚕变态发育的详细过程。通过对家蚕基因组数据库进行筛选,共在家蚕中鉴定到13种组织蛋白酶,并对这13种组织蛋白酶的基本信息和表达模式进行了分析。另外,利用家蚕基因芯片数据和荧光定量PCR分析,鉴定编号为BGIBMGA004622的基因为卵巢特异表达的组织蛋白酶L亚家族基因。该基因全长1 209 bp,编码402个氨基酸。经过序列分析,该酶与其他物种的组织蛋白酶L具有较高的同源性,其活性位点高度保守,且与鳞翅目的组织蛋白酶L在进化上聚为一支。同时,对该基因进行克隆并原核表达,结果显示重组蛋白以包涵体的形式表达。定量PCR结果显示,该酶在蛹发育初期表达量逐渐升高,至蛹3 d达到最高值,推测其可能参与卵巢与卵母细胞的发育过程。