Engineering hepatocyte growth factor fragments with high stability and activity as Met receptor agonists and antagonists
Engineering hepatocyte growth factor fragments with high stability and activity as Met receptor agonists and antagonists
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DOI：
10.1073/pnas.1102561108
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发表时间：
2011-08-09
影响因子：
11.1
通讯作者：
Cochran, Jennifer R.
中科院分区：
文献类型：
--
作者：
Jones, Douglas S., II;Tsai, Ping-Chuan;Cochran, Jennifer R.
Met受体酪氨酸激酶及其配体肝细胞生长因子(HGF)在调节肿瘤进展和组织再生中发挥重要作用。HGF的N端和第一Kringle结构域(NK1)包含一种自然产生的剪接变异体，它保留了激活Met受体的能力。然而，NK1是一种弱激动剂，且相对不稳定，限制了其治疗潜力。在这里，我们设计了具有改善的生化和生物物理特性的NK1突变体，其功能是Met受体激动剂或拮抗剂。我们首先利用定向进化的方法设计了NK1，以提高稳定性和重组表达产量。从我们的文库筛选中分离的NK1变异体由于NK1同源二聚化界面的突变而起到弱Met受体拮抗剂的作用。我们引入了点突变来恢复NK1同源二聚化界面以创建激动型配体，或者进一步破坏该界面以创建更有效的拮抗剂。经过合理设计的拮抗剂表现出高达约摄氏度的融化温度，比来自野生型NK1的拮抗剂提高了15摄氏度，表达产量提高了约40倍。接下来，我们通过引入N-末端的半胱氨酸残基来创建二硫键连接的NK1同源二聚体。与野生型NK1相比，这些共价二聚体的激动剂活性几乎提高了一个数量级，接近全长HGF的活性。此外，由激动型或拮抗型单体亚基形成的共价NK1二聚体也具有类似的活性，进一步表明NK1二聚体介导了激动型活性。这些经过改造的NK1蛋白有望用于治疗开发，并将成为进一步探索Met受体激活决定因素的有用工具。
The Met receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) play an important role in mediating both tumor progression and tissue regeneration. The N-terminal and first Kringle domains (NK1) of HGF comprise a naturally occurring splice variant that retains the ability to activate the Met receptor. However, NK1 is a weak agonist and is relatively unstable, limiting its therapeutic potential. Here, we engineered NK1 mutants with improved biochemical and biophysical properties that function as Met receptor agonists or antagonists. We first engineered NK1 for increased stability and recombinant expression yield using directed evolution. The NK1 variants isolated from our library screens acted as weak Met receptor antagonists due to a mutation at the NK1 homodimerization interface. We introduced point mutations that restored this NK1 homodimerization interface to create an agonistic ligand, or that further disrupted this interface to create more effective antagonists. The rationally engineered antagonists exhibited melting temperatures up to approximately 64 degrees C, a 15 degrees C improvement over antagonists derived from wild-type NK1, and approximately 40-fold improvement in expression yield. Next, we created disulfide-linked NK1 homodimers through introduction of an N-terminal cysteine residue. These covalent dimers exhibited nearly an order of magnitude improved agonistic activity compared to wild-type NK1, approaching the activity of full-length HGF. Moreover, covalent NK1 dimers formed from agonistic or antagonistic monomeric subunits elicited similar activity, further signifying that NK1 dimerization mediates agonistic activity. These engineered NK1 proteins are promising candidates for therapeutic development and will be useful tools for further exploring determinants of Met receptor activation.