Construction of a mutated pro-nerve growth factor resistant to degradation and suitable for biophysical and cellular utilization

Abstract

Precursor of nerve growth factor (proNGF) has been found to be proapoptotic in several cell types and mediates its effects by binding to p75 neurotrophin receptor (p75 NTR ) and sortilin. The proNGF molecule is processed by proteases at three dibasic sites found in the pro domain to form mature NGF (termed herein as sites 1, 2, and 3 from the proNGF N terminus). Of these processing sites, site 3, adjacent to the N terminus of mature NGF, was thought to be the major site responsible for processing of proNGF to mature NGF. We found that mutating this major processing site (site 3) resulted in a form of proNGF that was only partially stable. On introducing additional mutations in the pro domain at the other two dibasic sites, we found the stability of proNGF to increase significantly. Here we describe the construction, expression, and purification of this more stable proNGF molecule. The two consecutive basic residues at each of the three sites were mutated to neutral alanine residues. Expression was performed in stably transfected Sf21 insect cells. Purification involved strong cationexchange chromatography and N60 immunoaffinity column chromatography. The construct with all three sites mutated (termed proNGF123) gave all proNGF with no mature NGF and was not cleaved by three proconvertases (furin, PACE-4, and PC-2) known to proteolyze proneurotrophins in vivo. This stable proNGF molecule demonstrated proapoptotic activity on rat pheocytochroma PC12 cells, PC12nnr cells, C6 glioblastoma cells, and RN22 schwannoma cells.p75 receptor ͉ Sf21 stable cell line ͉ Trk receptor ͉ neurotrophin ͉ apoptosis

A Pro-Nerve Growth Factor (proNGF) and NGF Binding Protein, α2-Macroglobulin, Differentially Regulates p75 and TrkA Receptors and Is Relevant to Neurodegeneration Ex Vivo and In Vivo

“…Furin treatment cleaved proNGF (32 kDa), causing a time-dependent increase in the mature NGF subunits (26 kDa and 13 kDa) as digestion products. The remaining proNGF that was not processed after furin treatment was very heterogeneous (28 to 33 kDa), as reported by other researchers (31,32). In contrast, proNGF plus ␣ 2 M (e.g., within an ␣ 2 MproNGF complex) was fully protected from furin cleavage, and no products were detected even after a 90-min incubation.…”

“…Digestion assays were performed using 1 unit of furin (New England BioLabs), as described previously (31,32). Furin (1 U) was added to 100 ng of proNGF with or without ␣ 2 M or a BSA control.…”

Activation of pro‐BDNF by the pericellular serine protease plasmin

“…It is notable that mutation of a single cleavage site abolishes processing of BDNF, whereas for NGF it is necessary to mutate three sites in order to prevent processing [21]. Consistent with this, expression of RKAA-mtNGF in S2 cells generates multiple partially processed forms (Gray and Ellis, unpublished data).…”

Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation

“…Secreted AP and AP-proNGF were produced in HEK293T cells and normalized for molar concentrations, as described in Flanagan and Cheng (18). ProNGF123 was prepared and purified as described (19). To produce biotinylated proNGF, cDNA encoding proNGF with the mutated furin cleavage site was subcloned into pEGFPN1-secVSVg-Bgl-BAP, a plasmid encoding a biotin acceptor peptide (BAP) at the amino terminus.…”