Fikes J D, Barkocy-Gallagher G A, Klapper D G, Bassford P J
Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill 27599-7290.
J Biol Chem. 1990 Feb 25;265(6):3417-23.
Comparative analyses of a number of secretory proteins processed by eukaryotic and prokaryotic signal peptidases have identified a strongly conserved feature regarding the residues positioned -3 and -1 relative to the cleavage site. These 2 residues of the signal peptide are thought to constitute a recognition site for the processing enzyme and are usually amino acids with small, neutral side chains. It was shown previously that the substitution of aspartic acid for alanine at -3 of the Escherichia coli maltose-binding protein (MBP) signal peptide blocked maturation by signal peptidase I but had no noticeable effect or MBP translocation across the cytoplasmic membrane of its biological activity. This identified an excellent system in which to undertake a detailed investigation of the structural requirements and limitations for the cleavage site. In vitro mutagenesis was used to generate 14 different amino acid substitutions at -3 and 13 different amino acid substitutions at -1 of the MBP signal peptide. The maturation of the mutant precursor species expressed in vivo was examined. Overall, the results obtained agreed fairly well with statistically derived models of signal peptidase I specificity, except that cysteine was found to permit efficient processing when present at either -3 and -1, and threonine at -1 resulted in inefficient processing. Interestingly, it was found that substitutions at -1 which blocked processing at the normal cleavage site redirected processing, with varying efficiencies, to an alternate site in the signal peptide represented by the Ala-X-Ala sequence at positions -5 to -3. The substitution of aspartic acid for alanine at -5 blocked processing at this alternate site but not the normal site. The amino acids occupying the -5 and -3 positions in many other prokaryotic signal peptides also have the potential for constituting alternate processing sites. This appears to represent another example of redundant information contained within the signal peptide.
对一些经真核和原核信号肽酶加工的分泌蛋白进行的比较分析，已确定了相对于切割位点 -3 和 -1 位置的残基存在一个高度保守的特征。信号肽的这两个残基被认为构成了加工酶的识别位点，通常是具有小的中性侧链的氨基酸。先前已表明，将大肠杆菌麦芽糖结合蛋白（MBP）信号肽 -3 位的丙氨酸替换为天冬氨酸会阻断信号肽酶 I 的成熟作用，但对 MBP 跨细胞质膜的转运或其生物活性没有明显影响。这确定了一个极好的系统，可用于详细研究切割位点的结构要求和限制。体外诱变用于在 MBP 信号肽的 -3 位产生 14 种不同的氨基酸替换，在 -1 位产生 13 种不同的氨基酸替换。检测了体内表达的突变前体物种的成熟情况。总体而言，获得的结果与根据统计学推导的信号肽酶 I 特异性模型相当吻合，只是发现当半胱氨酸存在于 -3 或 -1 位时可允许有效加工，而 -1 位的苏氨酸会导致加工效率低下。有趣的是，发现 -1 位的替换若阻断正常切割位点的加工，则会以不同效率将加工重定向至信号肽中由 -5 至 -3 位的 Ala-X-Ala 序列代表的另一个位点。将 -5 位的丙氨酸替换为天冬氨酸会阻断该替代位点的加工，但不影响正常位点的加工。许多其他原核信号肽中占据 -5 和 -3 位的氨基酸也有可能构成替代加工位点。这似乎代表了信号肽中所含冗余信息的另一个例子。
