Studies on structural stability, receptor binding, and biological activity of nerve growth factor: Importance of the C-terminus and a core residue

Nerve growth factor (NGF) is found as a dimer of two processed 118 amino-acid polypeptides in the mouse submaxillary gland although the sequence predicts two additional amino-acids arg-gly at positions 119–120. Previous studies from our laboratory show that recombinantly produced mouse beta NGF (full-length 120 amino-acids denoted here as rwtNGF) has reduced receptor binding as well as neurite outgrowth-promoting effect in pheochromocytoma cells, which are restored to wild-type levels with cleavage of the C-terminal dipeptide (Luo and Neet, 1992). To investigate this region further, I have studied two mutant molecules with longer C-termini: 120H₆ (which has a hexa-histidine tail after position 120) and 120ADDN (with amino-acids ala-asp-asp-asn added at positions 120–124). The 120H₆ mutant showed reduced receptor binding, which was restored by cleavage of the tail after arg-118. Correspondingly, this C-terminal extension reduced neurite outgrowth function of the molecule in defined medium. This was restored by cleaving the extension and by exposure to serum medium. Serum medium did not physically cleave the C-termini, affecting the mutants through a conformational mechanism. The more negatively charged extension in 120ADDN, in contrast, did not seem to affect receptor binding or biological activity. However, the active conformation of this mutation was extremely unstable and lost biological activity rapidly with 1–2 freeze-thaw cycles. To differentiate between local (direct receptor binding of the C-terminal extension) or more long-range conformational effects observed with 120H₆, 120ADDN, and rwtNGF, I have studied the molecule, W21L, which contains the C-terminal dipeptide found in rwtNGF but has an additional point mutation at a central location: trp-21 changed to leucine. The W21L mutant had reduced receptor binding as well as greatly reduced bioactivity, however, our studies could not detect any restoration with cleavage of the C-terminal extension. Structural stability of mutant molecules before C-terminal cleavage was studied by urea gradient gel electrophoresis which showed no gross compromisation of conformational stability as compared to mouse beta NGF from the submaxillary gland. We conclude that the structure of the C-terminus is an important determinant of NGF functionality although the determination of the exact nature of the effect will require more study.