Robbi M, Beaufay H, Octave J N
Laboratoire de Chimie Physiologique, Université Catholique de Louvain, Brussels, Belgium.
Biochem J. 1990 Jul 15;269(2):451-8. doi: 10.1042/bj2690451.
A commercial rat liver cDNA library in lambda gt11 was screened with a rabbit antiserum to native pI 6.1 esterase (ES-10). The inserts of the immunoreactive clones were short (0.9-1.1 kbp). One of these was used as a probe to rescreen the library, yielding 30 clones, two of which contained relatively long (approx. 1.9 kbp) and widely overlapping cDNA inserts. They did not contain the first two nucleotide residues of the initiator codon, nor the 5'-end untranslated portion of the mRNA. These were derived from a home-made rat liver cDNA library in lambda gt11, screened with an oligonucleotide corresponding to the 5'-end of the already known cDNA sequence. The nucleotide sequence consists of 48 bp of 5'-end non-coding region, 1695 bp of coding region and 212 bp of 3'-end non-coding region including a 20 bp poly(A) tail. The signal peptide and the mature protein subunit are 18 and 547 residues long respectively. Tyr is confirmed as N-terminal residue. The predicted amino acid sequence is highly similar to those of rabbit liver esterase forms 1 (77% identity) and 2 (56% identity), determined by protein sequencing [Korza & Ozols (1988) J. Biol. Chem. 263, 3486-3495; Ozols (1989) J. Biol. Chem. 264, 12533-12545]. The three enzymes share the Ser and His residues presumed to be part of the active site, four Cys residues and a high proportion of charged side chains at their C-terminus. The C-terminal tetrapeptides of the three esterases (-HVEL, -HIEL and -HTEL for pI 6.1 and forms 1 and 2 esterases respectively) are reminiscent of, but not identical with, the localization signal identified in other proteins of the endoplasmic-reticulum lumen (-KDEL in animal cells [Munro & Pelham (1987) Cell 48, 899-907]; -HDEL in yeast [Pelham, Hardwick & Lewis (1988) EMBO J. 7, 1757-1762]). We still lack direct evidence to decide whether or not these C-terminal tetrapeptides commit esterases to reside in the endoplasmic reticulum. In that case the antepenultimate residue (D, V, I or T) would be only weakly stringent, and some sequences primed by H instead of K would be recognized in animal as well as in yeast cells.
用兔抗天然pI 6.1酯酶（ES-10）抗血清筛选λgt11载体中的商业大鼠肝脏cDNA文库。免疫反应性克隆的插入片段较短（0.9 - 1.1 kbp）。其中一个用作探针重新筛选文库，得到30个克隆，其中两个包含相对较长（约1.9 kbp）且广泛重叠的cDNA插入片段。它们不包含起始密码子的前两个核苷酸残基，也不包含mRNA的5'端非翻译部分。这些片段来自自制的λgt11载体大鼠肝脏cDNA文库，用与已知cDNA序列5'端对应的寡核苷酸进行筛选。核苷酸序列由5'端非编码区的48 bp、编码区的1695 bp和3'端非编码区的212 bp组成，包括一个20 bp的聚腺苷酸尾。信号肽和成熟蛋白亚基分别由18个和547个残基组成。酪氨酸被确认为N端残基。预测的氨基酸序列与通过蛋白质测序确定的兔肝脏酯酶1型（77%同一性）和2型（56%同一性）高度相似[科尔扎和奥佐尔斯（1988年）《生物化学杂志》263卷，3486 - 3495页；奥佐尔斯（1989年）《生物化学杂志》264卷，12533 - 12545页]。这三种酶共享推测为活性位点一部分的丝氨酸和组氨酸残基、四个半胱氨酸残基以及它们C端高比例的带电荷侧链。三种酯酶的C端四肽（pI 6.1酯酶以及酯酶1型和2型分别为 -HVEL、-HIEL和 -HTEL）让人联想到但不同于在内质网腔其他蛋白质中鉴定出的定位信号（动物细胞中的 -KDEL [蒙罗和佩勒姆（1987年）《细胞》48卷，899 - 907页]；酵母中的 -HDEL [佩勒姆、哈德威克和刘易斯（1988年）《欧洲分子生物学组织杂志》7卷，1757 - 1762页]）。我们仍然缺乏直接证据来确定这些C端四肽是否使酯酶定位于内质网。在这种情况下，倒数第二个残基（D、V、I或T）的严格性较低，并且在动物细胞和酵母细胞中，一些由H而非K引发的序列也会被识别。
