Heinzelman Pete, Schoborg Jennifer A, Jewett Michael C
Department of Chemical, Biological and Materials Engineering, University of Oklahoma, Sarkeys Energy Center, 100 East Boyd Street, Room T-301, Norman, OK 73019, USA
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208-3120, USA.
Protein Eng Des Sel. 2015 Oct;28(10):481-9. doi: 10.1093/protein/gzv022. Epub 2015 Apr 15.
Systemic injection of granulocyte colony-stimulating factor (G-CSF) has yielded encouraging results in treating Alzheimer's Disease (AD) and other central nervous system (CNS) disorders. Making G-CSF a viable AD therapeutic will, however, require increasing G-CSF's ability to stimulate neurons within the brain. This objective could be realized by increasing transcytosis of G-CSF across the blood brain barrier (BBB). An established correlation between G-CSF receptor (G-CSFR) binding pH responsiveness and increased recycling of G-CSF to the cell exterior after endocytosis motivated development of G-CSF variants with highly pH responsive G-CSFR binding affinities. These variants will be used in future validation of our hypothesis that increased BBB transcytosis can enhance G-CSF therapeutic efficacy. Flow cytometric screening of a yeast-displayed library in which G-CSF/G-CSFR interface residues were mutated to histidine yielded a G-CSF triple His mutant (L109H/D110H/Q120H) with highly pH responsive binding affinity. This variant's KD, measured by surface plasmon resonance (SPR), increases ∼20-fold as pH decreases from 7.4 to below histidine's pKa of ∼6.0; an increase 2-fold greater than for previously reported G-CSF His mutants. Cell-free protein synthesis (CFPS) enabled expression and purification of soluble, bioactive G-CSF triple His variant protein, an outcome inaccessible via Escherichia coli inclusion body refolding. This purification and bioactivity validation will enable future identification of correlations between pH responsiveness and transcytosis in BBB cell culture model and animal experiments. Furthermore, the library screening and CFPS methods employed here could be applied to developing other pH responsive hematopoietic or neurotrophic factors for treating CNS disorders.
全身注射粒细胞集落刺激因子（G-CSF）在治疗阿尔茨海默病（AD）和其他中枢神经系统（CNS）疾病方面已取得了令人鼓舞的成果。然而，要使G-CSF成为一种可行的AD治疗药物，需要提高其刺激脑内神经元的能力。这一目标可以通过增加G-CSF穿过血脑屏障（BBB）的转胞吞作用来实现。G-CSF受体（G-CSFR）结合pH反应性与内吞作用后G-CSF向细胞外的再循环增加之间已建立的相关性，促使人们开发具有高度pH反应性G-CSFR结合亲和力的G-CSF变体。这些变体将用于未来验证我们的假设，即增加BBB转胞吞作用可以提高G-CSF的治疗效果。对一个酵母展示文库进行流式细胞术筛选，其中G-CSF/G-CSFR界面残基被突变为组氨酸，得到了一个具有高度pH反应性结合亲和力的G-CSF三重组氨酸突变体（L109H/D110H/Q120H）。通过表面等离子体共振（SPR）测量，该变体的解离常数（KD）在pH从7.4降至组氨酸的pKa约6.0以下时增加约20倍；这一增加幅度比先前报道的G-CSF组氨酸突变体大2倍。无细胞蛋白质合成（CFPS）能够表达和纯化可溶性、生物活性的G-CSF三重组氨酸变体蛋白，这是通过大肠杆菌包涵体复性无法实现的结果。这种纯化和生物活性验证将有助于未来在BBB细胞培养模型和动物实验中确定pH反应性与转胞吞作用之间的相关性。此外，这里采用的文库筛选和CFPS方法可应用于开发其他用于治疗CNS疾病的pH反应性造血或神经营养因子。
