Velásquez André C, Nomura Kinya, Cooper Max D, Herrin Brantley R, He Sheng Yang
DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824 USA.
Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322 USA.
Plant Methods. 2017 Apr 19;13:29. doi: 10.1186/s13007-017-0180-8. eCollection 2017.
The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein-protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies.
In this study, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs. HopM1-specific VLRBs (VLR) were expressed in the cytosol, the -Golgi network, and the apoplast. Expression of VLR was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLR was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLR specifically interacted with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB.
In the future, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest , with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.
靶向和操纵基于蛋白质的细胞过程的能力将加速植物研究；然而，特异性和选择性靶向植物表达蛋白质的技术仍处于起步阶段。富含亮氨酸重复序列（LRR）是普遍存在的蛋白质结构域，参与介导蛋白质-蛋白质相互作用。LRR赋予无颌脊椎动物产生的高度多样化的可变淋巴细胞受体（VLR）抗体（包括VLRA、VLRB和VLRC类型）结合特异性，这些抗体作为有颌脊椎动物基于免疫球蛋白的抗体的功能等效物。
在本研究中，通过免疫七鳃鳗并利用酵母表面展示筛选高亲和力的VLRB，开发了靶向植物病原体效应蛋白HopM1的VLRB。靶向HopM1的特异性VLRB（VLR）在细胞质、高尔基体网络和质外体中表达。当蛋白质定位于有利于二硫键形成的氧化环境时（当VLR不定位于细胞质时），VLR的表达更高，因为二硫键对于VLR的正确折叠是必需的。VLR与HopM1特异性相互作用，但与无关的细菌效应蛋白不相互作用，而HopM1与非特异性VLRB不相互作用。
未来，VLR可作为灵活的模块来结合感兴趣的蛋白质或碳水化合物，通过直接结合其靶标并抑制其作用，或通过创建具有新特异性的嵌合蛋白（其中内源性LRR结构域被VLR中的结构域取代），具有广泛的应用可能性。
