Kornacker M G, Faucher D, Pugsley A P
Unité de Génétique Moléculaire (Centre National de la Recherche Scientifique UA1149, Institut Pasteur, Paris, France.
J Biol Chem. 1991 Jul 25;266(21):13842-8.
Site-directed mutagenesis was used to construct three mutant derivatives of the extracellular, cell surface lipoprotein pullulanase (PulA) in which the normally fatty acylated cysteine of the signal peptide-bearing precursor was replaced by other amino acids. When produced in Escherichia coli expressing all genes required for pullulanase secretion, approximately 90% of the PulA derivatives persisted as cell-associated precursors, indicating inefficient signal peptide processing. Processed (intermediate-sized) forms of the two derivatives that were studied in detail were found to result from proteolytic cleavage at different sites within the signal peptide. Both were further processed to smaller polypeptides by cleavage at an undetermined site that is presumably close to their C termini. The intermediate-sized pullulanase derived from prepullulanase in which Cys+1 had been replaced by Leu and Gly-1 by Glu (PulA:C1L/G-1E) appeared rapidly, was apparently entirely extracellular, and accounted for approximately 10% of synthesized PulA. Prolonged incubation did not result in further conversion of the precursor to the intermediate form, and the precursor remained anchored to the cytoplasmic membrane. The smaller processed form was also found extracellularly. The active form of the extracellular enzyme was monomeric, which is again in contrast to the fatty acylated, wild-type enzyme. Taken together, these results indicate that replacement of Cys+1 of prePulA eliminates processing by lipoprotein signal peptidase and does not permit processing by leader peptidase, but allows inefficient, aberrant processing by an unknown peptidase and immediate secretion of the resulting polypeptide, which retains most of its signal peptide. Processing and secretion only occur when the pullulanase secretion functions are expressed.
定点诱变用于构建细胞外、细胞表面支链淀粉酶（PulA）的三种突变衍生物，其中携带信号肽的前体中正常脂肪酰化的半胱氨酸被其他氨基酸取代。当在表达支链淀粉酶分泌所需所有基因的大肠杆菌中产生时，约90%的PulA衍生物以细胞相关前体的形式存在，表明信号肽加工效率低下。对两种详细研究的衍生物的加工（中等大小）形式进行分析，发现它们是由信号肽内不同位点的蛋白水解切割产生的。两者都通过在一个可能靠近其C末端的未确定位点的切割进一步加工成较小的多肽。源自前支链淀粉酶的中等大小的支链淀粉酶，其中Cys+1被Leu取代，Gly-1被Glu取代（PulA:C1L/G-1E）迅速出现，显然完全位于细胞外，约占合成的PulA的10%。长时间孵育不会导致前体进一步转化为中间形式，并且前体仍锚定在细胞质膜上。较小的加工形式也在细胞外被发现。细胞外酶的活性形式是单体，这再次与脂肪酰化的野生型酶形成对比。综上所述，这些结果表明，前PulA的Cys+1被取代消除了脂蛋白信号肽酶的加工，不允许前导肽酶进行加工，但允许一种未知肽酶进行低效、异常的加工，并立即分泌产生的多肽，该多肽保留了其大部分信号肽。只有当表达支链淀粉酶分泌功能时才会发生加工和分泌。
