Secreted Human Amyloid Precursor Protein Binds Semaphorin 3a and Prevents Semaphorin-Induced Growth Cone Collapse

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-β peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα695 (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα695 binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα695 inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (Kd≤8·10−9 M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins

Semaphorin 3A specifically binds sAPPα.

<p>(A) Wells in a 96-well plate were coated with either 50 ng sAPPα₆₉₅ (APP), 1 µg BSA or 50 ng Semaphorin 3A (Sema3A), blocked with 1% BSA and overlayed with different concentrations of Sema3A or sAPPα₆₉₅ (as indicated below the lanes). After washing, binding was determined by probing with anti-Sema3A antibody. The graph shows concentration-dependent binding of Sema3A to sAPPα₆₉₅ by densitometric quantification of the immunodots using NIH Image J. Bars represent averages of three independent experiments performed in triplicate each. The blot illustrates a representative experiment. (B) Wells in a 96-well plate were coated with 100 ng sAPPα₆₉₅ (APP), blocked with 1% BSA and incubated with increasing volumes of conditioned medium containing alkaline phosphatase-conjugated Semaphorin 3A (Sema3A-AP). After washing, binding was determined by measuring alkaline phosphatase activity. Bars represent averages of three independent experiments performed in triplicate. (C) Streptavidin-coated beads were incubated with biotinylated sAPPα₆₉₅ (bAPP) and Sema3A-AP-conditioned medium or control (non-transfected) medium. After washing, binding was evaluated by measuring alkaline phosphatase activity associated with the beads. Bars represent averages of three independent experiments.</p

sAPPα modulates the biological activity of Sema3A.

<p>Growth cone morphology was examined in control chick DRG explants (A,E) and in explants challenged for 30 minutes with 0.8 nM Sema3A (B,F), Sema3A+75 nM sAPPα₆₉₅ (C,G) or Sema3A+sAPPα₆₉₅+75 nM each of peptides ARSHPAM and LTASLLI (D,H). After fixation and neurofilament immunostaining, growth cone morphology was examined using fluorescence (upper panels) or phase contrast microscopy (lower panels). Scale bars: 10 µm. Representative images (A–H) illustrate on average two growth cones, but more than 700 growth cones were evaluated in each experimental condition. Panel I shows results of quantitative analysis of the percentage of collapsed growth cones (as determined from phase contrast images) in each experimental condition. Bars correspond to means ± SEM of different ganglia. Each experimental condition was replicated 3–6 times in independent experiments using different DRG cultures. Asterisks represent statistically significant differences (**p<0.01; ***p<0.001; ANOVA followed by Bonferroni post-hoc test).</p

sAPPα-binding peptides selected by phage display are homologous to members of the human semaphorin family.

<p>(A) Sequence alignment of the LRSHPLG and TFASVMT peptides with members of the human semaphorin family; identical residues are shown in red and conservative amino acid replacements are in blue. Sequence alignment was performed using ClustALL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022857#pone.0022857-Larkin1" target="_blank">[56]</a> (B) Ribbon representation of the structure of the receptor binding module of Semaphorin 3A. The amino acid sequences homologous to the LRSHPLG (blue; ARSHPAM) and TFASVMT (red; LTASLLI) peptides are highlighted. Residues involved in receptor specificity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022857#pone.0022857-Koppel1" target="_blank">[39]</a> are shown in yellow and red. The figure was generated using RasMol and the atomic coordinates for Sema3A (Protein Data Bank accession code 1Q47; ref. 40).</p