We cloned Aplysia netrin-1 and found that it is 47% identical and 60% similar to human netrin-1 (by comparison, C. elegans netrin-1 is 44% identical and 59% similar to human netrin-1).
We found that monocytes pretreated with aspirin displayed reduced motility towards the chemokine CCL2 compared with untreated cells, this effect being similar to that exerted by recombinant human netrin-1, and again this was abolished by UNC5b-Fc.
The LN and EGF domains of human netrin-1 relevant for DCC-binding were inserted into the AVEXIS bait vector.
To investigate netrin-1 mediated translation, 250 ng/ml recombinant Fc human netrin-1 (#ALX-522-455; Enzo life sciences, Farmingdale, NY) in L15 media or functional blocking anti-DCC (#OP45; Milipore, Billerica, MA) was applied to cultures for 24 hr and then washed with L15 media.
Cultures were incubated with 250 ng/ml recombinant Fc human netrin-1 for 24 hr.
The human netrin-1 gene is localized at 17p13-p12 [ 15] and encodes 604 amino acid protein of 70 84 kDa.
While we used recombinant human netrin-1 and function-blocking antibodies against human DCC, we are confident that these reagents reflect the endogenous functions of netrin-1 and DCC in Aplysia.
Aplysia Netrin-1 (ApNetrin-1, accession #KM218335) was 47% identical and 60% conserved with human Netrin-1, and Aplysia DCC (ApDCC, accession # KM218336) was 31% identical and 47% conserved with human DCC.
Further characterization of these Trio knockout mice revealed that Trio transduces signaling from the chemoattractant netrin-1 through binding to the netrin-1 receptor DCC (deleted in colorectal cancer), thus guiding the outgrowth and projection of commissure axons of cortical neurons [ 32].
We further characterized this interaction, narrowed down the binding interface, and demonstrated that Draxin competes with Netrin receptors for binding to Netrin-1.
