School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom.
Biopharmaceutical Development, MedImmune LLC, Gaithersburg, MD 20878, USA.
Biochim Biophys Acta Mol Cell Res. 2017 Jan;1864(1):202-208. doi: 10.1016/j.bbamcr.2016.10.018. Epub 2016 Oct 29.
The Tat system preferentially transports correctly folded proteins across the bacterial membrane although little is known of the proofreading mechanism. Most research has focused on TatABC systems from Gram-negative bacteria, especially Escherichia coli, and much less is known of the TatAC-type systems from Gram-positive organisms. We have previously shown that the Bacillus subtilis TatAdCd system is functional in an E. coli tat null background and able to transport TorA-GFP and native TorA (TMAO reductase); here, we examined its ability to transport other proteins bearing a TorA signal sequence. We show that whereas E. coli TatABC transports a wide range of biotherapeutics including human growth hormone, interferon α2b, a VH domain protein and 2 different scFvs, TatAdCd transports the scFvs but completely rejects the other proteins. The system also rejects two native E. coli substrates, NrfC and FhuD. Moreover, we have shown that TatABC will transport a wide range of folded scFv variants with the surface altered to incorporate multiple salt bridges, charged residues (5 glutamate, lysine or arginine), or hydrophobic residues (up to 6 leucines). In contrast, TatAdCd completely rejects many of these variants including those with 5 or 6 added Leu residues. The combined data show that the TatABC and TatAdCd systems have very different substrate selectivities, with the TatAdCd system displaying an extreme level of selectivity when compared to the E. coli system. The data also provide a preliminary suggestion that TatAdCd may not tolerate substrates that contain surface domains with a level of hydrophobicity above a certain threshold.
塔特（Tat）系统优先将正确折叠的蛋白质穿过细菌膜运输，尽管对其校对机制知之甚少。大多数研究都集中在革兰氏阴性菌（尤其是大肠杆菌）的 TatABC 系统上，而对革兰氏阳性菌的 TatAC 型系统则知之甚少。我们之前已经表明，枯草芽孢杆菌 TatAdCd 系统在大肠杆菌 tat 缺失背景下是有功能的，并且能够转运 TorA-GFP 和天然 TorA（氧化三甲胺还原酶）；在这里，我们研究了它转运带有 TorA 信号序列的其他蛋白质的能力。我们发现，虽然大肠杆菌 TatABC 可以转运多种生物治疗药物，包括人生长激素、干扰素 α2b、VH 结构域蛋白和 2 种不同的 scFv，但 TatAdCd 只能转运 scFv，而完全排斥其他蛋白质。该系统还排斥两种天然的大肠杆菌底物 NrfC 和 FhuD。此外，我们已经表明，TatABC 可以转运广泛的折叠 scFv 变体，其表面被改变以形成多个盐桥、带电荷的残基（5 个谷氨酸、赖氨酸或精氨酸）或疏水性残基（多达 6 个亮氨酸）。相比之下，TatAdCd 完全排斥许多这些变体，包括带有 5 个或 6 个添加亮氨酸残基的变体。综合数据表明，TatABC 和 TatAdCd 系统具有非常不同的底物选择性，与大肠杆菌系统相比，TatAdCd 系统表现出极高的选择性。该数据还初步表明，TatAdCd 可能无法耐受含有表面域的底物，这些表面域的疏水性超过一定的阈值。
