Barkocy-Gallagher G A, Bassford P J
Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill 27599-7290.
J Biol Chem. 1992 Jan 15;267(2):1231-8.
The residues occupying the -3 and -1 positions relative to the cleavage site of secretory precursor proteins are usually amino acids with small, neutral side chains that are thought to constitute the recognition site for the processing enzyme, signal peptidase. No restrictions have been established for residues positioned +1 to the cleavage site, although there have been several indications that mutant precursor proteins with a proline at +1 cannot be processed by Escherichia coli signal peptidase I (also called leader peptidase). A maltose-binding protein (MBP) species with proline at +1, designated MBP27-P, was translocated efficiently but not processed when expressed in E. coli cells. Unexpectedly, induced expression of MBP27-P was found to have an adverse effect on the processing kinetics of five different nonlipoprotein precursors analyzed, but not precursor Lpp (the major outer membrane lipoprotein) processed by a different enzyme, signal peptidase II. Cell growth also was inhibited following induction of MBP27-P synthesis. Substitutions in the MBP27-P signal peptide that blocked MBP translocation across the cytoplasmic membrane and, hence, access to the processing enzyme or that altered the signal peptidase I recognition site at position -1 restored both normal growth and processing of other precursors. Since overproduction of signal peptidase I also restored normal growth and processing to cells expressing unaltered MBP27-P, it was concluded that precursor MBP27-P interferes with the activity of the processing enzyme, probably by competing as a noncleavable substrate for the enzyme's active site. Thus, although signal peptidase I, like many other proteases, is unable to cleave an X-Pro bond, a proline at +1 does not prevent the enzyme from recognizing the normal processing site. When the RBP signal peptide was substituted for the MBP signal peptide of MBP27-P, the resultant hybrid protein was processed somewhat inefficiently at an alternate cleavage site and elicited a much reduced effect on cell growth and signal peptidase I activity. Although the MBP signal peptide also has an alternate cleavage site, the different properties of the RBP and MBP signal peptides with regard to the substitution of proline at +1 may be related to their respective secondary structures in the processing site region.
相对于分泌性前体蛋白切割位点而言，位于-3和-1位置的残基通常是具有小的中性侧链的氨基酸，这些氨基酸被认为构成了加工酶信号肽酶的识别位点。对于位于切割位点+1位置的残基尚未确定有任何限制，尽管有若干迹象表明，在+1位置带有脯氨酸的突变前体蛋白不能被大肠杆菌信号肽酶I（也称为前导肽酶）加工。一种在+1位置带有脯氨酸的麦芽糖结合蛋白（MBP），命名为MBP27-P，在大肠杆菌细胞中表达时能有效地转运但不能被加工。出乎意料的是，发现MBP27-P的诱导表达对所分析的五种不同的非脂蛋白前体的加工动力学有不利影响，但对由不同的酶信号肽酶II加工的前体Lpp（主要外膜脂蛋白）没有影响。在诱导合成MBP27-P后，细胞生长也受到抑制。MBP27-P信号肽中的取代阻止了MBP跨细胞质膜的转运，因此无法接触到加工酶，或者改变了-1位置的信号肽酶I识别位点，这恢复了其他前体的正常生长和加工。由于信号肽酶I的过量产生也使表达未改变的MBP27-P的细胞恢复了正常生长和加工，因此得出结论，前体MBP27-P可能通过作为一种不可切割的底物竞争酶的活性位点来干扰加工酶的活性。因此，尽管信号肽酶I与许多其他蛋白酶一样，不能切割X-Pro键，但+1位置的脯氨酸并不妨碍该酶识别正常的加工位点。当用RBP信号肽取代MBP27-P的MBP信号肽时，所得的杂合蛋白在一个交替的切割位点加工效率有点低，并且对细胞生长和信号肽酶I活性的影响大大降低。尽管MBP信号肽也有一个交替的切割位点，但RBP和MBP信号肽在+1位置脯氨酸取代方面的不同特性可能与它们在加工位点区域各自的二级结构有关。
