Interaction of antibodies to synthetic peptides of proNGF with in vitro synthesized NGF precursors

Abstract

Sera raised against three synthetic peptides that reproduce sequences of the pro-nerve powth factor (proNGF) protein were tested in immunoprecipitation experiments using in vitro translation products of SP6-directed NGF mRNA in a rabbit reticulocyte lysate. The interaction of these antibodies with bacterially synthesixed chimeric preproNGF was also examined. Digestion of the translation products by the y-subunit generated the 22 and 18 kDa intermediates. A predominant 13 kDa intermediate was obtained after digestion of translation products in wheat germ extract. This is shown to be the N-terminal peptide by immunoprecipitation with an anti-peptide serum. These antibodies may be used to detect NGF precursor cleavage products in vivo.

Characterization of nerve growth factor precursor protein expression by human prostate stromal cells: A role in selective neurotrophin stimulation of prostate epithelial cell growth

“…While both the NGF␥ subunit and trypsin appeared to cause a small increase in the electrophoretic mobility of the NGF precursor, these proteases failed to cleave the precursor to a mature form, while resulting in an apparent decrease in precursor. This suggests a different folding of precursor, since it has been observed that NGF␥ and trypsin degrade the mature NGF␤-moiety of in vitro NGF precursor translation products, suggested to be a consequence of improper folding [6][7][8]23]. While neither NGF␥ nor trypsin appeared to cleave the stromal NGF precursor to NGF␤, it is possible that another enzyme could cleave the protein to the mature form.…”

“…A similar effect of epitope masking and/or inability to recognize altered conformational epitopes may account for the inability of anti-NGF␤ to recognize intermediate forms of pro-NGF. In addition, differences in relative abundance of NGF precursors between sample preparations have been described [7] that could also account for differences in propeptide antibody recognition of precursor proteins. On infrequent occasions, NGF immunoblots of hPS preparations have also identified a protein with approximate M r of 18 kDa (data not shown), consistent with differences in the relative abundance of NGF precursors described previously [7].…”

“…In addition, differences in relative abundance of NGF precursors between sample preparations have been described [7] that could also account for differences in propeptide antibody recognition of precursor proteins. On infrequent occasions, NGF immunoblots of hPS preparations have also identified a protein with approximate M r of 18 kDa (data not shown), consistent with differences in the relative abundance of NGF precursors described previously [7]. In any event, it seems clear that human prostatic stromal cells express the 35-kDa and 27-kDa precursor forms of the prepro-NGF gene product, and that the processing of this gene product can produce the 22-kDa form of pro-NGF, but that processing is incomplete since the 13-kDa mature form of NGF␤ was never observed.…”

“…Hence, these results suggest that the 35-kDa NGF␤ immunoreactive protein in hPS is the larger precursor form of the NGF gene product. In order to investigate NGF precursors in hPS, two additional antibodies raised against biosynthetic peptides corresponding to −40 to −3 (L38 peptide) and −71 to −43 (N4 peptide) of the NGF precursor protein [7,28] were utilized for Western blot analysis of hPS. Immunoblot analysis of hPS with anti-N4 antibody identified two immunoreactive bands with molecular weights of approximately 35 kDa and 27 kDa, whereas, as expected, the anti-N4 antibody was not immunoreactive with the 13-kDa mature form of purified NGF␤, or in hPS.…”

“…NGF and related neurotrophin family members are all synthesized as precursor proteins which contain dibasic amino-acid proteolytic cleavage sites which may generate several intermediate precursor forms as well as the mature neurotrophin proteins [3][4][5]. Expression of NGF and other neurotrophins in vitro, as well as by various cell types transfected with neurotrophin genes, demonstrated the production of precursors which could be cleaved to mature forms by proteolysis with specific enzymes [6,7]. In particular, NGF␥ has been observed to be capable of processing NGF precursors to various molecular weight forms as well as to mature 13-kDa NGF␤ [8].…”