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