The expression system affects the binding affinity between p75NTR and proNGF

• Published in: Biochemistry and biophysics reports 2024-07, Vol.38, p.101702-101702, Article 101702
• Main Authors: Hino, Mami, Nakanishi, Masayuki, Nomoto, Hiroshi
• Format: Article
• Language: English
• Subjects: Binding affinity; Glycoform; p75NTR; proNGF; Protein expression system
• ISSN: 2405-5808; 2405-5808
• DOI: 10.1016/j.bbrep.2024.101702

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. •Recombinant proNGF and p75NTR are expressed in bacterial, insect, and mammalian cells.•Insect p75NTR shows a higher binding affinity to proNGFs than mammalian p75NTR.•Insect p75NTR binds insect proNGF with the highest affinity and bacteria proNGF lowest.•Mammalian p75NTR shows no preference for proNGF from different sources.•Glycan structures of these proteins are different, and glycans likely affect the affinity.