F3/contactin‐related proteins in Helix pomatia nervous tissue (HCRPs): Distribution and function in neurite growth and neurotransmitter release

Abstract

By using antibodies against mouse F3/contactin, we found immunologically related glycoproteins expressed in the nervous tissue of the snail Helix pomatia. Helix contactin-related proteins (HCRPs) include different molecules ranging in size from 90 to 240 kD. Clones isolated from a cDNA expression library allowed us to demonstrate that these proteins are translated from a unique 6.3-kb mRNA, suggesting that their heterogeneity depends on posttranslational processing. This is supported by the results of endoglycosidase F treatment, which indicate that the highmolecular-weight components are glycosylation variants of the 90-kD chain. In vivo and in cultures, HCRPs antibodies label neuronal soma and neurite extensions, giving the appearance of both cytoplasmic and cell surface immunostaining. On the other hand, no expression is found on nonneural tissues. Functionally, HCRPs are involved in neurite growth control and appear to modulate neurotransmitter release, as indicated by the inhibiting effects of specific antibodies on both functions. These data allow the definition of HCRPs glycoproteins as growth-promoting molecules, suggesting that they play a role in neurite development and presynaptic terminal maturation in the invertebrate nervous system. Keywords axonal glycoproteins; neurite growth; neurotransmitter release; F3/contactinIn vertebrates, axonal growth and pathfinding control depend on mechanisms involving a large set of neuronal surface glycoproteins, which belong to distinct families (Dickson, 2002;De Castro, 2003;Wen and Zheng 2006;Maness and Schachner, 2007). In particular, molecules built by the association of immunoglobulin type C2 and fibronectin type III (FNIII) domains play a relevant role in basic events of neuronal differentiation as axonal growth, fasciculation, and pathfinding (Brummendorf and Lemmon, 2001), a role also played by several extracellular matrix components (Galtrey and Fawcett, 2007). Depending on the mechanism of membrane association, axonal surface glycoproteins may be classified

The mouse F3/contactin glycoprotein

“…In addition related molecular species have been recently demonstrated in the nervous tissue of the terrestrial snail Helix Pomatia. 83,84 Together, these data support a highly conserved role for these molecules along phylogenesis.…”

Characterization and role ofHelixcontactin‐related proteins in culturedHelix pomatianeurons

“…Concentrated CM was then analyzed by Western blotting on 10% polyacrylamide gels as described by Milanese et al (2008). PF3 primary antiserum or preimmune serum (both 1:1,000 dilution in saturation buffer) was used, followed by HRPlabelled goat anti-rabbit IgG and the chemiluminescent ECL system (GE Healthcare, Piscataway, NJ).…”

“…Immunohistochemical staining was performed as described by Milanese et al (2008). PF3 antibody and the secondary biotinylated goat anti-rabbit serum (Vector Laboratories, Burlingame, CA) were used at 1:1,000 and 2 lg/ml dilutions, respectively.…”

“…In a previous study, mouse F3/contactin antibodyinhibition approaches revealed that HCRPs share part of these functions, including the control of neuronal adhesion to the substrate and of axonal growth; in addition, these molecules play a role in neurotransmitter release (Milanese et al, 2008). In the same study, HCRPs were found to include different glycosylated peptides, ranging in size from 90 to 240 kD.…”

“…In particular, in Aplysia californica and Helix pomatia nervous tissues, specific hints about the function of several adhesive glycoproteins have been obtained, including the Aplysia hortologue of the neural cell adhesion molecule (ApCAM; Mayford et al, 1992;Schacher et al, 2000;Ghirardi et al, 2001) and a family of F3/contactin-related glycoproteins called ''Helix contactin-related proteins'' (HCRPs; Milanese et al, 2008).…”