摘要
为发现新的辅脂酶样家族成员,通过EST拼接获得1个新的人类辅脂酶样蛋白编码基因hCLPSL3(human colipase-like 3);同时,通过同源性分析,对小鼠(Mus musculus)、大鼠(Rattus norvegicus)同源基因Clpsl3的完整编码区进行分析和预测。利用RT-PCR技术以人细胞系cDNA为模板成功扩增到hCLPSL3的2个转录变异体,即hCLPSL3-v1和-v2,前者包含完整的开放阅读框架(ORF),后者ORF被提前终止密码子(premature stop codon,PTC)中断;hCLPSL3-v1包含159个氨基酸,含有1个典型的信号肽,2个高度同源的内部序列重复区以及2个不完整的辅脂酶样结构域。通过真核表达载体构建,在人293T细胞中以过表达方式证实了hCLPSL3-v1能够分泌。通过RT-PCR技术,在小鼠肾、结肠和脾脏组织中各获得1个Clpsl3转录变异体序列,分别称为mCLPSL3-v1、-v2和-v3,其中mCLPSL3-v1包含完整的编码区,编码产物含161个氨基酸,而mCLPSL3-v2和-v3的ORF均被PTC中断;从大鼠结肠和小肠组织中克隆到大鼠Clpsl3的cDNA序列,即rCLPSL3,其编码162个氨基酸。此外,CLPSL3在哺乳动物中广泛存在,基因结构相似,均含有高度保守的18个半胱氨酸,推测与二硫键形成有关。这些工作为CLPSL3的功能研究奠定了基础。
Although ten years have passed since the draft sequence of the human genome was reported in 2001, the accurate number of human protein-encoding genes is still uncertain. According to the latest release of H-InvDB （release 6.2）, 34511 proteiwencoding genes were annotated and most of them were function-unknown. There is still a long road ahead to identify the whole human genes and their functions. During our previous genome-wide analysis of single-block EST sequences with polyadenylation sites, we found a lot of ESTs that represented potentially novel un identified genes or splice variants. In the present study, we focused on the cloning and identification of a novel cDNA sequence represented by the EST AV653338 with the encoding product containing two incomplete colipase like domains, when the electronic prolongation by EST contig was performed. By using mixed cDNAs from human cell lines as templates, we successfully cloned the novel encoding gene sequence, and revealed a novel human colipase-like protein-encoding gene named hCLPSL3 （human colipase-like 3） for there have been two other genes with protein products also containing colipase-like domains registered in the international nucleotide databases, C60RF126 （namely CLPSL1 here） and C60RF127 （namely CLPSL2 here）. Totally, two transcript variants were obtained, i.e. hCLPSL3-vl and -v2. Only hCLPSL3-vl contained a complete open reading frame （ORF）, encoding 159 amino acids, whereas the ORF of hCLPSL3-v2 was interrupted by PTC （premature stop codon） and it possibly was a substrate of nonsense-mediated mRNA decay. Human CLPSL3-vl contained a typical signal peptide （aa. 1-22） predicted by SignalP 4.0 Server, two internal sequence repeat regions （aa. 46-84 and aa. 88-125） with high homology, and two incomplete colipase-like domains （aa. 25- 68 and aa. 113-159） predicted by InterProScan and Motif Scan program, respectively. Using nested RT-PCR method for expression analysis in several cell lines （293T, HeLa, U2OS, HepG2, HCT116, A549, H1299, Jurkat, H520 and THP-1）, we found that hCLPSL3 was mainly expressed in 293T, U2OS, HCTll6 and THP- 1 cells. For lack of cDNAs, we did not perform the detection in human tissues. The typical signal peptide in CLPSL3 suggests that it probably is a secreted protein. Therefore, the recombinant eukaryotic expression vector of hCLPSL3-vl was constructed, and over-expressed in human 293T cells. As expected, human CLPSL3 was verified to be secreted when the supernatant was used for Western blot assay. By homology analysis, mouse and rat CLPSL3 homologous cDNAs were also predicted. Using RT-PCR method, three mouse CLPSL3 transcript variants （mCLPSL-vl, -v2 and -v3） were successfully cloned in the kidney （-vl）, colon （-v2） and＂ spleen （-v3） tissues, respectively. Only mCLPSL-vl contained a complete ORF encoding 161 amino acids, whereas the ORFs of both mCLPSL-v2 and -v3 were interrupted by PTCs. Rat CLPSL3 （rCLPSL3） cDNAs, encoding a product of 162 amino acids, were successfully cloned in both colon and small intestine tissues. However, rCLPSL3 was undetectable in rat pancreas. CLPSL3 was widely expressed and highly conserved in mammalian animals, such as Pan troglodytes, Equus caballus, Cavia porcellus, Loxodonta a fricana, Mus musculus and Rattus norvegicus, and showed very similar gene structures. Moreover, the CLPSL3 contained 18 highly conserved cysteines in all species, suggesting that it might relate to the disulfide bond formation. Colipase is a co-factor needed by pancreatic lipase for efficient dietary lipid hydrolysis. Because both mCLPSL and rCLPSL3 were detectable for expression in digestive tract, whether they play important roles in dietary lipid hydrolysis still needs further investigation. Our studies lay a foundation for future functional study of CLPSL3. In addition, all of the novel nucleotide sequences have been submitted to GenBank database with the accession numbers： JQ012741 （hCLPSL3-vl）, JQ012742 （hCLPSL3-v2）, JQ258939 （mCLPSL-vl）, JQ258940 （mCLPSL- v2）, JQ258941 （mCLPSL-v3） and JQ258942 （rCLPSL3）.
出处
《浙江大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2012年第5期575-584,共10页
Journal of Zhejiang University:Agriculture and Life Sciences
基金
北京市自然科学基金资助项目(5122016)
