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青岛文昌鱼(Branchiostoma belcheri tsingtaunese)GST和FREP基因的表达与功能
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摘要
文昌鱼(amphioxus)是介于无脊椎动物和脊椎动物之间的过渡类型,是现存的与脊椎动物祖先最接近的无脊椎动物,一直被认为是研究脊椎动物起源和进化的重要模式动物。文昌鱼基因的序列和表达模式已经被广泛用于不同物种之间的比较基因组学研究和发育同源性分析。本文从青岛文昌鱼(Branchiostoma belcheri tsingtaunese)肠cDNA文库中,分离了具有完整编码框的,与脊椎动物肝脏功能密切相关的谷胱苷肽转硫酶(GST)和纤维蛋白原相关蛋白(FREP)基因,并对它们的结构、进化、表达和功能进行了研究。
     我们在文昌鱼的肠cDNA文库中分离到了一个全长1032 bp的GST cDNA,它最长的开放阅读框编码一个227个氨基酸的蛋白,预测其分子量为26 kDa。BLASTp搜索结果显示该蛋白的N-端(3-79位残基)为GST_N结构域,C-端(120-204为残基)为GST_C结构域。用BbGST-1和其他14类胞质GSTs的氨基酸序列构建系统进化树,发现BbGST-1与多数水生生物(包括鱼类和绿藻)GSTs聚为一支,而与其它胞质GSTs明显分开。多序列比对结果显示BbGST-1与真鲷、黑呆头鱼、金头鲷、欧鲽、大嘴鲈鱼、牙鲆、斑马鱼和绿藻的GSTs分别有51.1%、48.9%、47.4%、48.4%、48.0%、47.6%、51.8%和43.7%的同源性,而与其他类的胞质GSTs同源性均低于27%。经分析,佛罗里达文昌鱼的基因组数据库中也存在GST的cDNA(和BbGST-1氨基酸序列同源性高达93%)和基因组DNA,表明BbGST-1在种内高度保守。基因组结构分析表明,文昌鱼GST基因与斑马鱼的假设GST,真鲷GSTR1和欧鲽GSTA1基因都具有6个外显子和5个内含子。这表明文昌鱼GST的基因组组成与其他几种鱼的GST一致。构建BbGST-1原核表达载体,转化大肠杆菌后诱导表达。纯化的重组蛋白对CDNB表现出相对高的活性(3.37±0.1 unit/mg),对EA表现出低的活性(0.41±0.01 unit/mg),而对DCNB, CHP, 4NBC和4HNE没有检测到活性。原位杂交显示BbGST-1转录本在文昌鱼的肝盲囊表达最丰富,在内柱、咽上沟、后肠、鳃和精巢有微弱表达,而在肌肉、脊索和神经管没有表达。用纯化的重组BbGST-1免疫小鼠制备抗血清,免疫组化实验进一步表明BbGST-1主要分布于肝盲囊、后肠、鳃和卵巢。所有这些表明文昌鱼GST-1属于一类新的GST——rho类,并且其表达具有组织特异性。同时这也显示在GST合成方面,文昌鱼的肝盲囊和脊椎动物的肝脏具有相似性,支持了文昌鱼肝盲囊和脊椎动物肝脏在功能上的等价性。
     我们还分离了一个编码纤维蛋白原相关蛋白(fibrinogen-related protein, FREP)的基因BbFREP。BbFREP全长1110 bp,它的开放阅读框编码一个286个氨基酸的蛋白,预测其分子量为32 kDa。推测BbFREP有一29个氨基酸残基的信号肽,并且在148位残基有一潜在的Asn-糖基化位点。BLASTp搜索显示,BbFREP在62-281位残基间有一FBG结构域。多序列比对分析表明,BbFREP的FBG和其他已知的FREPs有33.7-43.3%的同源性,它们都有4个保守的半胱氨酸残基。用这些FREPs的FBG结构域构建的进化树表明:BbFREP与人纤维蛋白原β和γ链聚为一支,而与其他FREPs相分开。实时定量PCR显示:在经过脂多糖(LPS)或磷壁酸(LTA)的刺激后,BbFREP的表达量会显著上升,峰值分别达到未处理组的38.8和21.4倍,这表明BbFREP可能为一免疫防御相关分子。通过毕赤酵母重组表达,获得纯化的BbFREP成熟肽,它可以特异性识别细菌表面的病原相关分子模式(PAMPs),包括LPS、肽聚糖(PGN)和LTA。另外重组蛋白对革兰氏阴性菌大肠杆菌和革兰氏阳性菌金黄色葡萄球菌表现出强的溶菌活性,扫描电子显微镜观察发现菌体表面受到破坏。Western blot结果显示,BbFREP也可以与大肠杆菌和金黄色葡萄球菌结合。原位杂交结果表明BbFREP主要在文昌鱼的肝盲囊和后肠表达,这也和其他FREP基因主要在脊椎动物肝脏表达结果一致。以上结果说明BbFREP作为一种模式识别受体起作用,并且可以通过与细菌表明的LPS、LTA和PGN结合,进而导致细菌溶解。另一方面,这些结果也支持了文昌鱼的肝盲囊与脊椎动物肝脏同源的观点,它们都是急性时相反应的主要组织。
Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. It is a well-known model organism widely used for interspecies comparative genome studies and developmental homology analysis. Here we obtain amphioxus glutathione S-transferase (BbGST-1) and fibrinogen-related protein (BbFREP) from gut cDNA library, and report the characterization, expression, phylogenetic analysis and functional characterization of them.
     The cDNA of BbGST-1 obtained from the gut cDNA library of amphioxus B. belcheri is 1032 bp long, and its longest open reading frame codes for a protein of 227 amino acids with a predicted molecular mass of approximately 26 kDa. The BLASTp searching at NCBI shows that the protein encoded by the cDNA has a GST_N domain at residues 3-79 and a GST_C domain at residues 120-204. To investigate the relationship between BbGST-1 and other GSTs, a phylogenetic tree is constructed using the amino acid sequence of BbGST-1 and that of other representative GSTs from 14 cytosolic classes, It is found that BbGST-1 formed a cluster together with the most GSTs from aquatic organisms including fish and alga, being obviously distinct from other cytosolic GST classes. Further comparison with piscine and algal GSTs reveals that BbGST-1 shared 51.1%, 48.9%, 47.4%, 48.4%, 48.0%, 47.6%, 51.8% and 43.7% identity to red sea bream, fathead minnow, gilthead sea bream, plaice, largemouth bass, flounder, zebrafish putative and green algal GSTs, respectively, while it had less than 27% identity to other GST classes. A search of the B. floridae genome revealed the presence of a Florida amphioxus GST-1 cDNA and its genomic DNA sequence. Sequence comparison demonstrated that BbGST-1 shared 93% similarity to the deduced protein encoded by Florida amphioxus GST-1 gene at the amino acid level, suggesting that GST-1 is highly conserved in intra-species. Analysis of the genomic structure exhibited that Florida amphioxus GST-1 gene consisted of six exons and five introns. It was notable that the zebrafish putative GST, rea sea bream GSTR1 and plaice GSTA1 genes all had six exons and five introns, indicating that amphioxus GST1 gene had an exon-intron organization similar to that of fish including zebrafish, sea bream and plaice. An expression vector including the entire open reading frame of BbGST-1 is constructed and transformed into E. coli. Recombinant protein is expressed and purified, shows a relatively high catalytic activity (3.37±0.1 unit/mg) toward CDNB and a moderate activity toward EA (0.41±0.01 unit/mg), but toward DCNB, CHP, 4NBC and 4HNE were not detectable. In situ hybridization histochemistry demonstrates that BbGST-1 transcript was most abundant in the hepatic caecum, and at a lower level present in the endostyle, epipharyngeal groove, hind-gut, gill, ovary and testis, while it is absent in the muscle, neural tube and notochord. This is further corroborated by the immunohistochemical staining using the mouse antisera against the purified recombinant BbGST-1, which shows that BbGST-1 is mainly localized in the hepatic diverticulum, hind-gut, gill and ovary. All these indicate that the amphioxus glutathione S-transferase belongs to a novel rho-class of glutathione S-transferases with a tissue-specific expression pattern. This also suggests that in respect of GST synthesis, the hepatic caecum in amphioxus is similar to the vertebrate liver, supporting the functional equivalence of amphioxus hepatic caecum to the vertebrate liver.
     The full length of BbFREP cDNA was 1110 bp with a longest open reading frame of 861 bp, which encoded a protein of 286 amino acids with a calculated molecular weight of about 32 kDa. The deduced protein also had a signal sequence of 29 residues and a potential Asn-linked glycosylation site at residual position 148. BLASTp searching at NCBI revealed that BbFREP had a FBG domain at residues 62-281 in its C-terminal region. Multiple sequence alignments revealed that the FBG domain of BbFREP shared 33.7-43.4% identity to that of known FREPs, and had all the four conserved cysteines. The phylogenetic tree constructed by neighbor-joining method using the conserved FBG domains of representative FREPs including BbFREP showed that BbFREP was clustered with human fibrinogenβandγchains, separating from other FREP members. Quantitative real time PCR revealed that the expression of BbFREP was significantly up-regulated following challenge with lipopolysaccharides (LPS) or lipoteichoic acid (LTA). These suggest that BbFREP is an immune defense-relevant molecule. The recombinant BbFREP expressed in pichia pastoris is able to specifically recognize the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces including LPS, peptidoglycan (PGN) and LTA, and displays strong bacteriolytic activities against both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus. BbFREP is also able to bind to both E. coli and S. aureus. In situ hybridization indicates that BbFREP is mainly expressed in the hepatic caecum and hind-gut, agreeing basically with the primary expression of vertebrate FREP genes in the liver. All these suggest that BbFREP can function as a pattern recognition receptor with a bacteriolytic activity via interaction with LPS, LTA and PGN. It also bolsters the notion that the hepatic caecum of amphioxus is equivalent to the vertebrate liver, acting as a major tissue in acute phase response.
引文
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