Construction and preliminary characterization of human recombinant proNGF-A variant
Introduction
Pro-nerve growth factor (proNGF) is the precursor of NGF and the prevalent form of the neurotrophin detectable in the nervous system (Bierl et al., 2005; Bierl and Isaacson, 2007; Fahnestock et al., 2001). Its conceivable relevance in the development and progression of neurological diseases is suggested by the increased amount of proNGF detected in both animal models of neurodegeneration and neurotraumas (Beattie et al., 2002; Soligo et al., 2015) and in human neurological diseases (Fahnestock et al., 2001). Despite these indications, there is still open debate about the biological role of proNGF in both physiological and pathological conditions.
Native proNGF is expressed, in mouse tissues, as two main different transcripts originating from alternative splicing and/or different promoters activation (Edwards et al., 1986; Racke et al., 1996). We recently produced evidences suggesting that two different protein variants, the long proNGF-A and the short proNGF-B, are produced in the rat brain and that their physiological ratio is altered by the development of neurodegenerative conditions (Protto et al., 2019; Soligo et al., 2019). This and other in vitro supportive evidences (Soligo et al., 2019), strengthen the hypothesis that different proNGF protein variants could play different roles in the mammalian nervous system.
After the first description of an active biological role of proNGF (Lee et al., 2001), as an inducer of apoptosis through the activation of p75NTR-sortilin receptor complex (Nykjaer et al., 2004), it became clear that the pro-neurotrophin may also activate pro-survival and differentiative downstream signaling in vitro, depending on the receptor/receptor complex challenged (Masoudi et al., 2009). It is worth noting that the majority of the works, addressing the bioactivity of proNGF, have been performed by means of recombinant proteins, carrying specific mutations aimed at interfering with the intracellular proteolysis and with subsequent maturation of proNGF in mNGF (Clewes et al., 2008; Lee et al., 2001; Masoudi et al., 2009; Pagadala et al., 2006; Seidah et al., 1996). Furthermore, both the transcripts have been expressed in prokaryotic and eukaryotic expression systems (Masoudi et al., 2009), but nobody addressed the possible correlation between the biological outcomes and the different expressed transcript, neither were investigated the possible structural differences between the protein variants originating from different cDNAs.
We recently produced in vitro data indicating that proNGF-A, but not proNGF-B, may have a pharmacological relevance, due to its neurotrophic activity and its resistance to extracellular proteases, when compared with mNGF (Soligo et al., 2019). In the present work, we aimed at studying aminoacid substitution in the proNGF peptide that could generate stable, recombinant human proNGF-A. Finally, we studied its metabolism and provided early in vitro pharmacological evidences, by exposing PC12 cells to conditioned media from transfected HeLa cells overexpressing human recombinant proNGF-A.
Section snippets
Human proNgf-A and proNgf-B qPCR design and amplification
To discriminate for the presence of proNgf-A and proNgf-B transcripts in human tissues, we designed specific qPCR assays, using the software Primer-BLAST, available online at http://www.ncbi.nlm.nih.gov/tools/primer-blast/(Figure S1). The primers used to amplify the proNgf-A mRNA spanned from 3044 to 3177 of the mRNA transcript XM_011541518 (Figure S1A-C; product length 133 bp), which is predicted on the basis of computational analysis of genomic sequences. The primers used for the
Analysis of proNgf's splicing variants in human brain
Although two alternative splicing variants have been reported in human (XM_011541518 and NM_002506, Figure S1A), the longer one (XM_011541518) was only predicted. To check for the presence of the two splicing variants in human tissues, we designed custom qPCR primers for the selective amplification of both the predicted proNgf-A and proNgf-B mRNAs (Figure S1B-C). The primers amplified the predicted cDNA fragments from human brain and synoviocytes cDNAs (Figure S1D), demonstrating that both the
Discussion
The existence of a NGF precursor protein (Berger and Shooter, 1977) and of the mRNA variants from which it is translated (Edwards et al., 1986) was demonstrated several years before the discovery of proNGF's specific biological activity (Lee et al., 2001). After further twenty years, a model of biological activity of proNGF, both in physiological conditions and during the development and progression of diseases, is still not defined. In vitro studies with recombinant mouse proNGF, made stable
Funding source
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
CRediT authorship contribution statement
Marzia Soligo: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Writing - review & editing, Visualization. Antonio Chiaretti: Resources. Eleonora Leotta: Investigation, Formal analysis. Elena Lardone: Investigation, Formal analysis. Chiara Boschelle: Investigation, Formal analysis. Elide Mantuano: Validation, Formal analysis. Liana Veneziano: Resources, Validation, Formal analysis. Luigi Manni: Conceptualization, Methodology, Investigation, Formal
Declaration of competing interest
None.
Acknowledgement
cDNAs from human synovial fibroblasts were graciously provided by Dr. Luisa Bracci-Laudiero, Ospedale Pediatrico Bambin Gesù, Rome, Italy. cDNAs obtained from human brain tissue were gifted by Policlinico Gemelli Foundation, Institute of Pediatrics.
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