Hino Mami, Nakanishi Masayuki, Nomoto Hiroshi
Laboratory of Biochemistry, School of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime, 790-8578, Japan.
Biochem Biophys Rep. 2024 Apr 1;38:101702. doi: 10.1016/j.bbrep.2024.101702. eCollection 2024 Jul.
ProNGF (nerve growth factor) is a precursor of NGF and a signaling peptide exerting opposite effects on neuronal cells, i.e., apoptotic or neuritogenic. The conflicting biological activity of proNGF depends on the relative levels of two membrane receptors, TrkA and p75NTR. The effect of proNGF depends on the expression levels of these receptor proteins and their affinity to proNGF. Since the affinity of proteins has been studied with various recombinant proteins, it is worth comparing the affinity of these proteins within one experiment with the same method. This study examined the affinity between a recombinant proNGF and p75NTR expressed in common systems: bacterial, insect, and mammalian cells. The extracellular domain of p75NTR expressed in the insect or mammalian systems bound to native mature NGF, with a higher affinity for the insect receptor. The uncleavable proNGF was expressed in the three systems and they showed neuritogenic activity in PC12 cells. These recombinant proteins were used to compare their binding affinity to p75NTR. The insect p75NTR showed a higher binding affinity to proNGF than the mammalian p75NTR. The insect p75NTR bound proNGF from the insect system with the highest affinity, then from the mammalian system, and the lowest from the bacterial system. Conversely, the mammalian p75NTR showed no such preference for proNGF. Because the recombinant proNGF and p75NTR from different expression systems are supposed to have the same amino acid sequences, these differences in the affinity depend likely on their post-translational modifications, most probably on their glycans. Each recombinant proNGF and p75NTR in various expression systems exhibited different mobilities on SDS-PAGE and reactivities with glycosidases and lectins.
前体神经生长因子（ProNGF）是神经生长因子（NGF）的前体，是一种对神经元细胞具有相反作用的信号肽，即具有凋亡或促神经突生长作用。ProNGF相互矛盾的生物学活性取决于两种膜受体TrkA和p75NTR的相对水平。ProNGF的作用取决于这些受体蛋白的表达水平及其对ProNGF的亲和力。由于已使用各种重组蛋白研究了蛋白质的亲和力，因此值得在同一实验中用相同方法比较这些蛋白质的亲和力。本研究检测了在常见系统（细菌、昆虫和哺乳动物细胞）中表达的重组ProNGF与p75NTR之间的亲和力。在昆虫或哺乳动物系统中表达的p75NTR的细胞外结构域与天然成熟NGF结合，对昆虫受体的亲和力更高。不可切割的ProNGF在这三种系统中表达，并且它们在PC12细胞中显示出促神经突生长活性。这些重组蛋白用于比较它们与p75NTR的结合亲和力。昆虫p75NTR对ProNGF的结合亲和力高于哺乳动物p75NTR。昆虫p75NTR对来自昆虫系统的ProNGF结合亲和力最高，其次是哺乳动物系统，对来自细菌系统的ProNGF结合亲和力最低。相反，哺乳动物p75NTR对ProNGF没有这种偏好。由于来自不同表达系统的重组ProNGF和p75NTR应该具有相同的氨基酸序列，因此这些亲和力差异可能取决于它们的翻译后修饰，很可能取决于它们的聚糖。各种表达系统中的每种重组ProNGF和p75NTR在SDS-PAGE上表现出不同的迁移率，并且与糖苷酶和凝集素具有不同的反应性。
