The pro‐sequence facilitates folding of human nerve growth factor fromEscherichia coliinclusion bodies

Abstract

Nerve growth factor (b-NGF), a neurotrophin required for the development and survival of specific neuronal populations, is translated as a prepro-protein in vivo. While the presequence mediates translocation into the endoplasmic reticulum, the function of the pro-peptide is so far unknown. As the pro-sequences of several proteins are known to promote folding of the mature part, the renaturation behaviour of recombinant human b-NGF pro-protein was compared to that of the mature form. Expression of rh-pro-NGF in Escherichia coli led to the formation of inclusion bodies (IBs). The presence of the covalently attached pro-sequence significantly increased the yield and rate of refolding with concomitant disulfide bond formation when compared to the in vitro refolding of mature NGF (rh-NGF). Physicochemical characterization revealed that rh-pro-NGF is a dimer. The pro-peptide could be removed by limited proteolysis with trypsin yielding biologically active, mature rh-NGF. Furthermore, rh-pro-NGF exhibited biological activity in the same concentration range as rh-NGF.

Biophysical Comparison of BMP-2, ProBMP-2, and the Free Pro-peptide Reveals Stabilization of the Pro-peptide by the Mature Growth Factor

“…In case of NGF, the pro-form has recently been attributed pro-apoptotic functions (13,15). In addition, we have previously demonstrated that the pro-peptide significantly contributes to in vitro oxidative folding (10,11). The original objective of the presented work was to show a similar role for the pro-peptide of BMP-2.…”

“…Because we had shown previously that the pro-form of NGF can also be cleaved in vitro by trypsin to obtain mature NGF (11), this protease was used to test maturation of proBMP-2 to BMP-2. Proteolysis with trypsin resulted in N-terminally truncated BMP-2 (Fig.…”

“…The presence of the pro-peptide can modulate or even change the biological activity of the respective cytokine. Several lines of evidence indicate that the pro-form of NGF induces pro-apoptotic signals (13,14), in contrast to NGF, which acts as a mitogen (11). It has recently been shown that the pro-peptide targets NGF to a newly described receptor protein, sortilin, that specifically binds the pro-form (15).…”

Biological Activity of Nerve Growth Factor Precursor Is Dependent upon Relative Levels of Its Receptors

“…Inclusion bodies were isolated from cell pellets, solubilized, and protein was refolded as described (27,33). Refolded protein was purified by fast protein liquid chromatography and eluted in 50 mM sodium phosphate, pH 7, 1 mM EDTA, 2 M NaCl.…”

“…In Alzheimer disease brain, retrograde transport from the cortex and hippocampus to basal forebrain cholinergic neurons is reduced as these neurons degenerate, with concomitant proNGF accumulation in the cortex and hippocampus (21,23). This suggested that proNGF mediates biological activity besides its prodomain function of promoting protein folding and regulation of neurotrophin secretion (25)(26)(27)(28). To study the role of proNGF protein in vitro, point mutations were inserted at the cleavage site used by furin, a proprotein convertase known to cleave proNGF (29), to minimize the conversion of proNGF to mature NGF.…”